Book of Abstracts

Regional Biophysics Conference 2007

21st – 25th August 2007

Balatonfüred Hungary

Editors

LÁSZLÓ ZIMÁNYI ZOLTÁN KÓTA BALÁZS SZALONTAI

Table of Contents PROGRAM.......................................................................................................................................................7 Tuesday, 21st August ..................................................................................................................................9 Wednesday, 22nd August ..........................................................................................................................10 Thursday, 23rd August..............................................................................................................................11 Friday, 24th August ..................................................................................................................................12 Saturday, 25th August...............................................................................................................................13 LECTURE ABSTRACTS ................................................................................................................................15 Tuesday, 21st August ...............................................................................................................................17 Wednesday, 22nd August..........................................................................................................................21 Thursday, 23rd August .............................................................................................................................34 Friday, 24th August ..................................................................................................................................52 Saturday, 25th August ..............................................................................................................................62 POSTER ABSTRACTS ...................................................................................................................................69 Macromolecular Structure and Function .................................................................................................71 Membranes, Receptors and Channels......................................................................................................93 Photobiophysics and Biological Energy Conversion.............................................................................113 Cells, Tissues, Organs and Complex Biological Systems .....................................................................125 Single Molecule Techniques, Nanotechnology, Microscopy and Imaging ...........................................135 Theoretical Biophysics, Bioinformatics and Computer Modelling .......................................................146 Miscellany .............................................................................................................................................158 AUTHOR INDEX .........................................................................................................................................165 LIST OF PARTICIPANTS ............................................................................................................................170

PROGRAM

TUESDAY 21ST AUGUST TUESDAY, 21ST AUGUST

12:00 – 16:00 Registration 16:00 – 16:30 Opening 16:30 – 18:30 Plenary.1 Chairman: Péter Závodszky 16:30 P.1.1 Alberto Diaspro: Tracking molecules and events in 3D using non linear excitation microscopy 17:10 P.1.2 Gerhard Schütz: Addressing plasma membrane structure at the nanometer length scale 17:50 P.1.3 Gábor Forgács: Relating biophysical properties across scales from the subcellular to the organ level 18:30

A.1 Hungarian Young Biophysicist Award Lecture Katalin Solymosi: The occurrence of NADPH:Protochlorophyllide oxidoreductase under natural conditions and the analysis of its activity in vitro

19:00 Get together party

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WEDNESDAY 22ND AUGUST WEDNESDAY, 22ND AUGUST

8:30 – 10:50 Plenary.2 Chairman: Alberto Diaspro 8:30 P.2.1 Karl Lohner: Role of lipid diversity for the selectivity of antimicrobial peptides 9:05 P.2.2 Mauro Dalla Serra: Permeabilizing activity of fuscopeptin, a lipodepsipeptide from Pseudomonas fuscovaginae, on natural and model membranes 9:40 P.2.3 Janez Štrancar: From local rotational conformational spaces to the protein structures 10:15 P.2.4 Péter Závodszky: Allostery - ligand dependent conformational dynamics in enzymes 10:50 – 11:30 Coffee break 11:30 – 13:00 Session a.1 Chairman: László Mátyus 11:30 Sa.1.1 János Szöllısi: Biophysics of ErbB2 signalling 12:00 Sa.1.2 Michaela Schwarzenbacher: Micropatterning of membrane proteins for screening molecular interactions in living cells – a case study on lck binding to CD4 12:30 Sa.1.3 Ales Iglič: Budding of biological membranes 11:30 – 13:00 Session b.1 Chairman: Ferenc Vonderviszt 11:30 Sb.1.1 Gábor Rákhely: Connection between the function of hydrogenase enzyme and photosynthetic electron transport in Thiocapsa roseopersicina BBS 12:00 Sb.1.2 Péter Maróti: Protein relaxation: case-history of bacterial reaction centre 12:30 Sb.1.3 Géza Groma: Light-induced ultrafast electron and proton motion in bacteriorhodopsin generates THz radiation 13:00 – 14:00 Lunch 14:00 – 15:00 Company presentations Chairman: György Lustyik 14:00 C.1 Arnaud Zoubir (Jobin Yvon): Raman microscopy: A powerful tool for probing biological systems 14:20 C.2 Róbert Szipıcs (R&D Ultrafast Lasers): Broadband and broadly tunable femtosecond pulse laser and parametric oscillators for applications in femtobiology 14:40 C.3 Paul Reinhard (Coherent): Advances in lasers for multiphoton excitation microscopy 17:00 – 19:00 Poster 1 19:00 – 20:00 Dinner 20:00 – Poster presentations

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Chairman: Lóránd Kelemen

THURSDAY 23RD AUGUST THURSDAY, 23RD AUGUST

8:30 – 10:50 Plenary.3 Chairman: Janez Štrancar 8:30 P.3.1 Pavle Andjus: Combating amyotrophic lateral sclerosis with biophysical means 9:05 P.3.2 Peter Pohl: Water transport through membrane channels 9:40 P.3.3 György Panyi: Coupling of activation and inactivation gating of Shaker K+ channels 10:15 P.3.4 Tibor Páli: Spin label EPR spectroscopy of the vacuolar proton-ATPase: structure, function and inhibitors 10:50 – 11:30 Coffee break 11:30 – 13:00 Session a.2 Chairman: Peter Pohl 11:30 Sa.2.1 Zoltán Varga: pH-dependent regulation of the inactivation of a lymphocyte potassium channel 12:00 Sa.2.2 László Csanády: Sulfonylurea receptors type 1 and 2A readily coassemble to form octameric KATP channels of mixed subunit composition 12:30 Sa.2.3 Marko Gosak: Intracellular Ca2+ signalling in hepatocytes after inhibition of plasma membrane Ca2+ efflux 11:30 – 13:00 Session b.2 Chairman: Miklós Nyitrai 11:30 Sb.2.1 Béla Suki: Elasticity and breakdown of soft biological tissues 12:00 Sb.2.2 Milan Brumen: Regulatory systems of signal transduction in airway smooth muscle contraction 12:30 Sb.2.3 Dénes Lırinczy: Thermodynamic parameters of actin under different agents and its possible role in the ATP hydrolysis cycle of muscle 13:00 – 14:00 Lunch 14:00 – 16:00 Session a.3 Chairman: László Zimányi 14:00 Sa.3.1 Goran Bačić: EPR spin-trapping of oxygen free radicals in chemical and biological systems: Reevaluation of the role of Fenton reaction 14:30 Sa.3.2 Béla Böddi: The physiological roles of the native protochlorophyllide complexes 15:00 Sa.3.3 Gyızı Garab: Thermo-optically induced reorganizations in light-harvesting antennae 15:30 Sa.3.4 István Hajdú: Adaptation of dehydrogenases to extreme environments 14:00 – 16:00 Session b.3 Chairman: Bojan Zagrovic 14:00 Sb.3.1 Csaba Bagyinka: How does the hydrogenase work? 14:30 Sb.3.2 László Smeller: Conformation and intermolecular interaction of proteins revealed by high pressure FTIR studies 15:00 Sb.3.3 Balázs Szalontai: Dynamics and interactions of lipids and proteins in biological membranes. An infrared spectroscopic study 15:30 Sb.3.4 Zoran Arsov: Amide hydrogen/deuterium exchange in sphingomyelin/cholesterol membranes studied by ATR-FTIR spectroscopy 16:00 Excursion, conference dinner

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FRIDAY, 24TH AUGUST FRIDAY, 24TH AUGUST

8:30 – 10:50 Plenary.4 Chairman: Pavle Andjus 8:30 P.4.1 Liviu Movileanu: Single-molecule stochastic sensing using nanopores 9:05 P.4.2 Paolo Facci: Electron transport through single redox metalloproteins: what scanning probe techniques can teach us 9:40 P.4.3 Iva Tolic-Norrelykke: Optical micromanipulations of the intracellular design 10:15 P.4.4 Dimitrije Stamenović: A molecular model of rheological behavior of living cells 10:50 – 11:30 Coffee break 11:30 – 13:00 Session a.4 Chairman: Paolo Facci 11:30 Sa.4.1 Miklós Kellermayer: Nanotechnology with amyloid fibrils 12:00 Sa.4.2 Christian Maurer: 3D movie of living fibroblasts 12:30 Sa.4.3 Sylvia Jeney: Observation of confined non-diffusive Brownian motion of an isolated particle 11:30 – 13:00 Session b.4 Chairperson: Marjeta Šentjurc 11:30 Sb.4.1 Raymond Sparrow: Synthetic biology and light-activated bio-molecular machines 12:00 Sb.4.2 Ales Omerzu: Optical transitions in M-DNA 12:30 Sb.4.3 Franci Merzel: An atomistic model of DNA: low frequency vibrations and basepair opening 13:00 – 14:00 Lunch 14:00 – 14:30 Round table: Future of the RBC 16:30 – 19:00 Poster 2 19:00 – 20:00 Dinner

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SATURDAY 25TH AUGUST SATURDAY, 25TH AUGUST

8:30 – 10:50 Plenary.5 Chairman: Dimitrije Stamenovic 8:30 P.5.1 István Simon: Protein structure is determined by the sequence via subset amino acid compositions 9:05 P.5.2 Bono Lučić: Modeling global properties of proteins 9:40 P.5.3 Bojan Zagrovic: Studying the extremes of biomolecular dynamics in silico: from protein folding to structure determination 10:15 P.5.4 Dusanka Janežič: Molecular modelling - protein-protein binding sites prediction 10:50 – 11:30 Coffee break 11:30 – 13:00 Plenary.6 Chairman: Pál Ormos 11:30 P.6.1 David Perahia: Global conformational changes, cavities, ligand binding and migration in proteins 12:00 P.6.2 Antonio Cupane: Tracking protein intramolecular motions in solution with timeresolved Wide Angle X-ray Scattering 12:30 P.6.3 Judit Fidy: Monitoring the subunit interface and conformational dynamics to unravel the mechanism of action of allosteric effectors in human haemoglobin 13:00 – 13:30 Closing

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LECTURE ABSTRACTS

LECTURE ABTRACTS TUESDAY, 21ST AUGUST Tuesday, 21st August

16:30- 17:10

Plenary Lecture 1.1. Tracking molecules and events in 3D using non-linear excitation microscopy. TESTA 1,3, I., GARRE 4, M ., PARAZZOLI 2, D., BAROZZI 2, S., MAZZA 1, D., FARETTA 2, M AND DIASPRO 1, 3, 4, 5, A. Department of Physics, University of Genoa, I-16146 Genoa, Italy. European Institute of Oncology, Department of Experimental Oncology, 20141 Milan, Italy. 3 LAMBS-MicroScoBio Research Center, University of Genoa, 16146 Genoa, Italy. 4 IFOM, Istituto FIRC di Oncologia Molecolare, 20139 Milan, Italy. 5 IBF-CNR, National Research Council, , I-16149 Genoa, Italy. 1 2

Green fluorescent protein (GFP) from Aqueorea Victoria [1] and its multicoloured variations on the theme are among the most routinely fluorescent tracers used for biological visualization [2]. The interest has grown in more precise localization studies of protein activity and movement within a cell and we could say that a new revolution started with the advent of photoactivatable fluorescent proteins [3]. Fluorescence of proteins effectively brought a “new light” in molecular and cellular biology studies [4], the “fluorescence toolbox” is growing [5] and steps towards macromolecular-scale resolution, using optical microscopes, are becoming reality. Within this pivotal scenario we focused on paGFP as photoactivatable fluorescent protein, and on the indispensable tools offered by confocal and two-photon microscopy [6, 7]. Particle tracking inside the cell largely benefits of the ability to spatially and temporally mark specific structures to follow their “signalling” over a “dark” background as made possible since the advent of the pa-GFP. In terms of spatial confinement of the photo activation process, the use of two-photon or even multiphoton excitation [8] provides several favourable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked [9]. The highly confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the 3D space. Even though single photon confocal laser scanning microscopy can efficiently modulate excitation power in planar sub-micron region, it fails to elicit the same control along the optical axis, being the excitation volume extended to the entire illumination cone of the objective. The ability to mark specific cells in living embryos by photo activating biomolecular markers can provide a unique tool in developmental biology studies to understand cell fate and mechanisms of differentiation. Non-linear spatially controlled protein photoconversion along the optical axis provides a unique possibility to dynamically identify single 3D structures and considering final 4D (x-y-z-t) processes. Moreover, the exploitation of non linear processes involved in the interaction between light and proteins has already lead to macromolecular-resolution levels keeping the advantages of using optical microscopy [10]. [1] O. Shimomura, J.Microscopy 217, 3 (2005). [2] Tsien, R. Y. Annual Review of Biochemistry, 67, 509 (1998). [3]Patterson, G. H.; Lippincott-Schwarz, J. Science, 297, 1873 (2002). [4] Diaspro A. Microsc Res Tech. 69, 149 (2006). [5] Giepmans BN, Adams SR, Ellisman MH, Tsien RY. Science. 312, 217 (2006). [6] Diaspro A. (ed) Confocal and Two-Photon Microscopy. Wiley-Liss (2001). [7] Diaspro A, Bianchini P, Vicidomini G, Faretta M, Ramoino P, Usai C. Biomed Eng Online. 5, 36 (2006). (http://www.biomedical-engineering-online.com/content/5/1/36) [8] Schneider M, Barozzi S, Testa I, Faretta M, Diaspro A. Biophys. J. 89, 1346 (2005). [9] Testa I, Parazzoli D, Barozzi S, Garrè M, Faretta M, Diaspro A. J. Microscopy, in press (2007) [10] Willig K.I., Kellner R.R., Medda R., Hein B., Jakobs S., Hell S.W. Nature Methods 3, 721 (2006). Acknowledgement This work was supported by IFOM (FIRC Institute of Molecular Oncology, Milan, Italy), by Fondazione Compagnia di San Paolo (Turin, Italy) and by AD PRIN2006-MiUR (Ministry of University and Research). The authors are indebted to George Patterson and Jennifer Lippincott-Schwartz for paGFP availability.

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LECTURE ABSTRACTS Tuesday, 21st August

17:10-17:50

Plenary Lecture 1.2. Addressing plasma membrane structure at the nanometer length scale WIESER, S.1, MOERTELMAIER, M.1, FUERTBAUER, E.2, STOCKINGER, H.2, SCHÜTZ, G.J.1 1 2

Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr.69, A-4040 Linz, Austria, [email protected] Department of Molecular Immunology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria

Current scientific research throughout the natural sciences aims at the exploration of the Nanocosm, the collectivity of structures with dimensions between 1 and 100nm. In the life sciences, the diversity of this Nanocosm attracts more and more researchers to the emerging field of Nanobiotechnology – biotechnology at or for the nanoscopic dimension. In this seminar, I will show examples how to obtain insights into the organization of the cellular Nanocosm by single molecule experiments. The plasma membrane structure affects interactions between membrane constituents by influencing their movements at the nanometer scale. We apply single molecule fluorescence microscopy to resolve the plasma membrane structure at a nanoscopic length-scale by employing the high precision for localizing biomolecules of down to 15nm. Minimum invasive labeling via fluorescent Fab fragments was sufficient to image the lateral diffusion of individual protein molecules on a sub-millisecond time scale. We applied this technology to study the motion of single glycosylphosphatidylinositol- (GPI-) anchored proteins in the plasma membrane of living cells 1. In contrast to results obtained by tracking gold-labeled membrane proteins, the single molecule fluorescence data reveal free Brownian motion of the proteins down to length scales of ~70nm, indicating no constitutive confinement zones. Acknowledgement This work was supported by the Austrian Science Fund (FWF), the Competence Center for Biomolecular ResearchVienna and by the GEN-AU project of the Austrian Federal Ministry for Education, Science and Culture. References [1] Wieser, S., Moertelmaier, M., Fürtbauer, E., Stockinger, H. & Schütz, G. J. Biophys. J. 92:3719-328 (2007).

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LECTURE ABTRACTS st

Tuesday, 21 August

17:50-18:30

Plenary Lecture 1.3. Relating biophysical properties across scales from the subcellular to the organ level GÁBOR FORGÁCS Department of Physics and Astronomy, University of Missouri, 420 Physics Building, Columbia, MO, 65211, USA

A distinguishing feature of a multicellular living system is that it operates at various scales and levels of organization, from the intracellular to organismal. Genes and molecules set up the conditions for physical mechanisms to act, in particular to shape the developing organism and establish its material characteristics. As development continues the changes brought about by the physical processes lead to changes in gene expression. It is through this coordinated interplay between genetic and generic (i.e. physical, chemical) regulation that the adult organism acquires its final structure and composition. It is natural to assume that in this multi-scale process the smaller defines the larger. In case of biophysical properties, in particular, those at the subcellular and cellular level are expected to give rise to those at the tissue level and beyond. Indeed, the physical properties of tissues vary greatly in physical properties: blood is liquid, bone is solid. In between these extremes lie most of the organs and tissues with typically intermediate viscoelastic properties. However, a blood cell is not the same as a liquid drop, a bone-forming cell (i.e. osteoblast) itself is not a solid and a single cardyomiocyte has entirely different physical properties than the mature heart. Very little is known at present on how tissue and organ level properties are related to cell and subcellular properties. We introduce a novel conceptual-theoretical-computational framework to address this fundamental question. The basis of our approach is a representation of a cell by a network of interacting ‘organelles’ (i.e. modules) with cell-specific properties. These cells form tissues through interactions with each other either by cell adhesion or through secreted substances. The tissues form organ structures through morphogenetic processes (e.g. fusion, compartmentalization) with characteristic shapes and physical properties. The theory incorporates morphogenetic processes by describing the movement of individual cells in the course of structure formation controlled by cellular and subcellular viscous and elastic properties. Our conceptual framework is motivated by tissue engineering applications where the biophysical properties of the engineered tissue or organ (e.g. blood vessel) are vital to physiological functions. We will validate the new formalism experimentally by applying it to specific morphogenetic processes such as tissue fusion and sorting. Acknowledgement This research is supported by a grant from the National Science Foundation FIBR-0526854.

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LECTURE ABSTRACTS Tuesday, 21st August

18:30-19:00

HUNGARIAN YOUNG BIOPHYSICIST AWARD LECTURE The Occurrence of NADPH:Protochlorophyllide Oxidoreductase under Natural Conditions and the Analysis of its Activity in vitro SOLYMOSI, K. Department of Plant Anatomy, Eötvös University, Pázmány P. s. 1/C., Budapest, 1117, Hungary, [email protected]

Chlorophyll (Chl) is essential for photosynthesis thus for all living organisms. Chl has an intense turnover in photosynthesising cells. Protochlorophyllide (Pchlide) is a Chl precursor, converted to chlorophyllide (Chlide) by a photoenzyme, NADPH:Protochlorophyllide OxidoReductase (POR) in angiosperms, i.e. the reaction is triggered by light and the enzyme cannot drive the reaction in the dark. As a consequence, darkgerminated angiosperm plants accumulate Pchlide. Pchlide pigments are present in different molecular microenvironments within the plastids; therefore they are organized in different complexes with various spectral properties. The activity of the POR enzyme is usually studied in dark-grown plant material, in which Chl biosynthesis is arrested and the reaction can be triggered by controlled illumination and can be easily followed. However, the question can be raised, whether dark-grown plants can be considered as adequate models for natural biosynthetic processes or represent a rather artificial system that does not occur in the nature. In order to answer this question, we studied different plant organs developing under natural light conditions, in which long-lasting or transient conditions for dark-growth could be supposed. Such structures are for instance cabbage heads [1], and buds of different woody plant species [2,3], in which the outer leaves envelop the inner tissues and the inner leaves. Due to this shading effect of the outer leaves, the innermost parts of these structures develop in complete darkness or at low light intensities. We have shown that Pchlide and POR accumulate in the inner leaves of cabbage heads [1] and also in the inner leaf primordia of several buds [2,3] developed under natural conditions. Similarly, Pchlide accumulation occurs and Chl biosynthesis is arrested in the pericarp-covered regions of low light-grown sunflower cotyledons [4]. This phenomenon is not so rare in the nature, as the pericarp often remains attached to the cotyledons 3-10 days after germination in the soil. When the pericarp detaches or the buds open, the Pchlide-Chlide transformation and Chl biosynthesis proceeds in a very similar way than in the artificially dark-germinated and illuminated seedlings. The above results indicate that the details of LPOR enzyme activity can be of general interest for plant biologists. Using high pressure fluorescence spectroscopy, we determined kinetical parameters and activation volumes linked to the phototransformation of Pchlide into Chlide in vitro in homogenates prepared from dark-grown wheat leaves [5]. The effect of high concentrations of Hg2+ on the stability and the activity of the enzyme complexes [6] and on the lipid environment of these enzyme complexes [7] has been also studied in order to understand the factors that are important in this unique light-dependent reaction mechanism. Acknowledgement I am grateful first of all to prof. Béla Böddi, my supervisor for supporting and encouraging my scientific career. I would also like to thank all co-authors without whom these results could not have been published. References [1] Solymosi K, Martinez K, Kristóf Z, Sundqvist C, Böddi B (2004) Physiol Plant 121: 520-529. [2] Solymosi K, Böddi B (2006) Tree Physiol 26: 1075-1085. [3] Solymosi K, Bóka K, Böddi B (2006) Tree Physiol 26: 1087-1096. [4] Solymosi K, Vitányi B, Hideg É, Böddi B (2007) Annals Bot 99: 857-867. [5] Solymosi K, Smeller L, Böddi B, Fidy J (2002) Biochim Biophys Acta 1554: 1-4. [6] Solymosi K, Lenti K, Myśliwa-Kurdziel B, Fidy J, Strzałka K, Böddi B (2004) Plant Biol 6: 358-368. [7] Solymosi K, Myśliwa-Kurdziel B, Bóka K, Strzałka K, Böddi B (2006) Plant Biology 8: 627-635

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LECTURE ABTRACTS WEDNESDAY, 22ND AUGUST Wednesday 22nd August

8:30-9:05

Plenary Lecture 2.1. Role of lipid diversity for the selectivity of antimicrobial peptides SEVCSIK,E., PABST,G. AND LOHNER, K. Institute of Biophysics and Nanostructure Research, Austrian Academy of Sciences, Schmiedlstrasse 6, Graz, A-8042, Austria, [email protected]

Nowadays, we face a world-wide rapid increase in pathogenic bacteria that are multi-resistant to commercially available antibiotics, while the number of novel antibiotics on the market declines. Thus, the emergence of multi-resistant bacteria poses a global health problem and urges for the development of novel antibiotics [1]. The discovery of antimicrobial peptides, effector molecules of the innate immune system, which confer a first line of defence against invading pathogens has opened new avenues for developing such drugs. These peptides, killing bacteria within minutes, can be found in every organism and display a wide spectrum of activity. Therefore there is an extensive effort to understand the mode of action to engineer peptides with an improved therapeutic index, i.e. high efficacy and target specificity [2]. As most of the antimicrobial peptides are believed to act via membrane disruption [3], we have performed biophysical studies using membrane model systems mimicking bacterial and mammalian cell membranes, which differ markedly in their membrane architecture in respect of complexity as well as lipid composition. These experiments have demonstrated that there are distinctive different mechanisms of bacterial killing by antimicrobial peptides. Therefore, the classical models of membrane perturbation (pore formation and carpet mechanism) are discussed and related to other mechanisms that may lead to membrane dysfunction such as formation of lipid-peptide domains, promotion of lipid chain interdigitation and membrane disruption by formation of micellar or non-lamellar lipid structures. Moreover, in certain cases one and the same peptide can exhibit different modes of membrane disintegration depending not only on lipid headgroup charge but also on hydrocarbon chain length. For example, the human multifunctional peptide LL-37 induces a peptide-associated quasi-interdigitated phase in negatively charged dipalmitoyl-phosphatidylglycerol, where the hydrocarbon chains are shielded from water by the peptide, but leads to a disintegration of the lamellar organization of zwitterionic dipalmitoyl-phosphatidylcholine into disk-like micelles. Such dual behavior was also observed for melittin and the antimicrobial frog skin peptide, PGLa, and can be attributed to a balance between electrostatic interactions, reflected in different penetration depths of the peptide, and hydrocarbon chain length. Thus, we suggest a phase diagram showing different macroscopic lipid phases as a function of the chemical nature of the phospholipid and peptide concentration, which is applicable to peptides localized parallel to the membrane surface. Acknowledgement We gratefully acknowledge financial support from the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich (P 15657) and by the Alois Sonnleitner Stiftung, Österreichische Akademie der Wissenschaften. References [1] Development of Novel Antimicrobial Agents: Emerging Strategies (Lohner K, ed.), Horizon Scientific Press, Wymondham, Norfolk, U.K. (2001). [2] Lohner, K., Blondelle, SE (2005) Comb. Chem. High Troughput Screening 8: 241-256. [3] Bechinger, B., Lohner K (2006) Biochim. Biophys. Acta 1758: 1529-1539.

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LECTURE ABSTRACTS Wednesday 22nd August

9:05-9:40

Plenary Lecture 2.2. Architecture of the pore formed on natural and model membranes by fuscopeptin, a lipodepsipeptide from Pseudomonas fuscovaginae CORAIOLA 1, M., PALETTI 1, R., FIORE 2, A., FOGLIANO 2, V. AND DALLA SERRA 1, M. 1 FBK 2 Dip.

& CNR-IBF – Unit at Trento, via alla Cascata 56/C, 38050 Povo (Trento), Italy, [email protected] Scienza degli Alimenti, Univ. di Napoli “Federico II”, Parco Gussone Edificio 84 80055 Portici, Napoli, Italy

Fuscopeptin, a lipodepsipeptide (LDP) produced by Pseudomonas fuscovaginae, is the causal agent of “sheath brown rot” disease of cultivated and wild Gramineae. This molecule is involved into the disease development. Here, we present a biophysical investigation of the interaction of fuscopeptin with natural and model membranes. Fuscopeptin was able to permeabilize erythrocytes and pure lipid membranes. The architecture of fuscopeptin pore has been further investigated by spectroscopic and electrophysiological techniques. From FTIR spectroscopy an increase in the helical content at expenses of random coil and betastructures was detected when the peptide enters the membrane from the buffer solution. Interestingly, the presence of both alpha and 3(10) helix could be clearly distinguished in the amide I. The fuscopeptin helical fractions were inserted into the membrane with an angle of about 30 degree with respect to the perpendicular to plane of the membrane, suggesting that fuscopeptin does not lay on the membrane surface but it is inserted into the lipid core. Fuscopeptin form pores into planar lipid bilayers: channel conductance is non-linear and depends on the membrane lipids composition. Channel opening is voltage dependent and is modulated by the electrical charge of the membrane. The cationic selectivity strongly increases if negative lipids are present into the membrane. Furthermore, fuscopeptin increases the transbilayer movement (flip-flop) of a fluorescently labelled lipid in liposomes: these evidences strongly support the toroidal architecture of the pore. Acknowledgement Sponsored by PAT Fondo Progetti (Project SyrTox)

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LECTURE ABTRACTS Wednesday 22nd August

9:40-10:15

Plenary Lecture 2.3. From local rotational conformational spaces to the protein structures ŠTRANCAR, J.1, KAVALENKA, A.1,2, STOPAR, D.3, ZIHERL, P.1,4, BELLE, B.5, FOURNEL, F.5, HEMMINGA, M.A.6 Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia, [email protected] Faculty of radio physics and electronics, Belarusian State University, 5, Kurchatova Street, Minsk, Belarus 3 Biotechnical faculty at University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia 4 Faculty of Mathematics and Physics at University of Ljubljana, Jadranska ulica 19, SI-1000 Ljubljana, Slovenia 5 Laboratoire de Bioénergétique et Ingénierie des Protéines , CNRS, 13402 Marseille Cedex, France 6 Wageningen University, Dreijenlaan 3, 6703 HA Wageningen , Netherlands 1 2

Recent developments in biological EPR and in the field of membrane protein structure determination methodology based on site-directed spin labeling EPR experiment, EPR spectra simulations, dHEO inverseproblem-solving, GHOST condensation and simulations of local rotational conformational spaces, enable us to tackle several problems in structural biology which cannot be directly addressed by high-resolution methods like X-ray crystallography and high-resolution NMR. Although this approach is recently developing for membrane protein structure determination, systems like very flexible water soluble (unstructured) proteins can also be addressed. We will present basic ideas experimentally detected with SDSL EPR experiments and appropriate spectral analysis that enables us the extraction of the restrictions of the local rotational motions. On the other hand, these restrictions can be simulated within an approach that combines the protein backbone modelling together by the modelling of the overlapping rotational conformational spaces of the protein side chains. By comparison of the experimentally detected and simulated local restrictions at the same time at various proteins sites, the global protein structure can be characterized. To improve the structural information, the effect of the lipid environment is taken into account in the case of membrane proteins. Similarly, in unstructured proteins the structure is improved by the low-resolution data from solution scattering experiment from protein complexes with other supramolecular structures. Furthermore we will discuss the effect of primary, secondary structure and lipids on sensitivity of this approach. Acknowledgement The authors acknowledge the financial support from the state budget by the Slovenian Research Agency (project P10060). References [1] STOPAR, David, ŠTRANCAR, Janez, SPRUIJT, Ruud B., HEMMINGA, Marcus A. Exploring the local conformational space of a membrane protein by site-directed spin labeling. J. chem. inf. mod., 2005, vol. 45, str. 1621-1627. [2] STOPAR, David, ŠTRANCAR, Janez, SPRUIJT, Ruud B., HEMMINGA, Marcus A. Motional restrictions of membrane proteins : a site-directed spin labeling study. Biophys. j., 2006, vol. 91, no. 9, str. 3341-3348. [3] ŠTRANCAR, Janez, KAVALENKA, Aleh, ZIHERL, Primož, STOPAR, David, HEMMINGA, Marcus A. Simulation of Rotational Conformational Spaces combined with SDSL ESR – a key for membrane protein structure determination. Submitted for publication.

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LECTURE ABSTRACTS Wednesday 22nd August

10:15-10:50

Plenary Lecture 2.4. Allostery - Ligand Dependent Conformational Dynamics in Enzymes ZÁVODSZKY P., HAJDÚ I., SZILÁGYI A. Institute of Enzymology, Hungarian Academy of Sciences, Karolina út 29, Budapest, H-1113, Hungary, [email protected]

There is a delicate balance between stabilizing and destabilizing interactions within the native 3D structure of proteins, giving rise to a marginal free energy of stabilization. This balance can easily be adjusted by one or a few replacements in the amino-acid sequence or by the binding of small molecular weight ligands to the protein. Enzymatic catalysis can be activated, inhibited and regulated by subtle environmental effects of low energy, such as change in pH or temperature or the binding of ligands. The unique nature of enzymes can be attributed to their diverse polyfunctional side chains and dynamic structure. It follows from the principles of statistical thermodynamics that proteins are dynamic and the protein matrix exhibits fluctuations (e.g. energy, volume) on a wide range of time scales, from femtoseconds to milliseconds. On the other hand, the structure of a protein molecule is cooperative and heterogeneous, therefore the fluctuations are concerted and unevenly distributed within the matrix. The question is whether there is a link between these conformational fluctuations and catalytic function. Allosteric signal transduction is a fundamental process used by proteins to propagate signals over long intramolecular distances. Originally, the concept of allostery or “different shape” was used to describe the coupling between conformational changes at distant sites, when the binding of one ligand alters the affinity of the enzyme toward a second one. We studied the ligand-induced conformational changes in the case of several enzymes (GAPDH, LDH, aldolase, IPMDH) and antibodies. The gross conformational changes as reflected by “shape” related parameters like intrinsic viscosity, radius of gyration, difference sedimentation, etc., were negligible, while significant changes in affinities for the second ligand were observed and differential scanning microcalorimetric measurements revealed changes in the conformational stabilities. Bulk hydrogen-deuterium exchange can detect changes in the overlapping local fluctuation pattern of proteins. The relaxation spectra describe the probability distribution of solvent accessibilities for buried peptide hydrogens. As an example, the binding of NAD coenzyme to GAPDH resulted in a shift of the H-D exchange relaxation spectra, reflecting a change in the fluctuation pattern of the molecule as a whole. Upon binding of monovalent hapten to induced homogeneous IgG molecules, we could trace signal propagation through the hinge region using the chromophoric probe 2,6-dichloromercury-4-nitrophenol, associated with an extended suppression of conformational changes throughout the whole 12-domain molecule, as revealed by bulk H-D exchange relaxation spectra. The binding of hapten also resulted in increased C1q binding at the Fc part of the IgG molecule, distant from the hapten binding site at the Fab region. Changes in flexibility upon ligand binding were observed in several allosteric proteins. Modulation of correlated conformational fluctuations can be a good means of energy transduction. From molecular dynamics simulations, entropy-order parameter profiles can be derived, and it is suggested that conformational entropy contributes to the fine-tuning of allosteric affinities. The alteration of correlated, overlapping local conformational fluctuations is likely to be the principal mechanism for allosteric communication between distant sites in a protein molecule.

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LECTURE ABTRACTS Wednesday 22nd August

11:30-12:00

Parallel Session a.1.1. Biophysics of ErbB2 signaling SZÖLLİSI 1,2, J., VEREB 1, G. AND NAGY 1, P. Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Nagyerdei krt 98, 4012 Debrecen, Hungary, [email protected] 2 Cell Biology and Signaling Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Nagyerdei krt 98, 4012 Debrecen, Hungary, 1

Function and integrity of cells are determined by the supramolecular organization of biomolecules at the cell surface or inside the cell. Molecular proximity and interactions of these molecules can be detected by specific techniques such as flow or image cytometric variations of fluorescence resonance energy transfer (FRET). Flow cytometric techniques offer the advantage of rapid analysis on a large number of cells (~105 cells in some minutes) with a high statistical accuracy and a possibility for analyzing heterogeneity at the population level. The new generation bench-top and research flow cytometers also offer the advantage of multiparameter analysis (increasing number of available laser-excited optical channels) and increased sensitivity owing to optical improvements in photon collection. Flow cytometry, however, does not provide any information about the spatial localization of fluorescent probes, but instead measures the fluorescence intensity averaged over each cell. In contrast, microscopic techniques provide a high spatial resolution: conventional fluorescence microscopies have a ~250 nm resolution limited by diffraction of the optics. Although microscopies have several further advantages in detecting molecular dynamics or kinetics of changes in the distribution or intensity of fluorescent probes, they suffer from a low statistical reliability, especially in the case of quantitative measurements. Thus, a combined application of flow and image cytometry in resolving particular biological questions can be a very powerful approach. In flow cytometry we applied fluorescent probes with longer wavelength excitation and multiple wavelength detection in the emission regions so that autofluorescence correction could be performed on a cell by cell basis in FRET analysis. These facts improved the accuracy of the FRET method and cells with low receptor expression. Combination of various forms of flow and image cytometric FRET methods revealed distinctive expression and association pattern of ErbB receptor tyrosine kinases on the surface of various cancer cell lines sensitive or resistant to trastuzumab (Herceptin®). Simultaneous application of image cytometric FRET methods based on donor and acceptor photobleaching provided a useful dual FRET approach revealing a unique coassociation pattern of integrins, CD44 and ErbB2 on the surface of tumor cells. By measuring the distances between various monoclonal antibody epitopes on ErbB2 molecules and the distances between epitopes and the cell membranes useful information was provided for positioning the extracellular domain in molecular modeling the nearly full length ErbB2 dimer. In this model favorable dimerization interactions were predicted for the extracellular, transmembrane and protein kinase domains, which may act in coordinated fashion in ErbB2 homodimerization, and also in heterodimers of ErbB2 with other members of ErbB family

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LECTURE ABSTRACTS Wednesday 22nd August

12:00-12:30

Parallel Session a.1.2. Micropatterning of Membrane Proteins for Screening Molecular Interactions in Living Cells – a case study on lck binding to CD4 SCHWARZENBACHER 1, M., KALTNEBRUNNER 1, M., BRAMESHUBER 1, M., HESCH 1, C., WEGHUBER 1, J., PASTER 2, W., STOCKINGER 2, H., HEISE 3, B., SONNLEITNER 4, A. AND SCHÜTZ 1, G.J. Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr.69, A-4040 Linz, Austria, [email protected] Department of Molecular Immunology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria 3 Department of Knowledge-based Mathematical Systems, Johannes Kepler University Linz, Altenberger Str.69, A-4040 Linz, Austria 4 Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Scharitzerstr.6-8, A-4020 Linz, Austria 1 2

We present here a method to identify and characterize interactions between an arbitrary fluorescence labeled protein (“prey”) and a membrane protein (“bait”) in living cells. Cells transfected with a fluorescent protein (FP-) fusion of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the exoplasmic domain of the bait; the fluorescence copatterning is used as readout for the interaction. We demonstrate the proof of principle by confirming the interaction between CD4, the major co-receptor in T cell activation, and lck, a leukocyte-specific protein tyrosine kinase essential for early T cell signaling. By probing the interaction directly in the living cells, environmental parameter were preserved; this enabled us to identify the critical role of lipid rafts for the interaction, assess the contribution of multiple lck interaction domains, and measure the lck-CD4 interaction lifetime. Acknowledgement This work was supported by the Austrian Science Fund (FWF), project numbers P15053, P15025 and Y250-B10, the Competence Center for Biomolecular Research-Vienna, and by the GEN-AU project of the Austrian Federal Ministry for Education, Science and Culture.

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LECTURE ABTRACTS Wednesday 22nd August

12:30-13:00

Parallel Session a.1.3. Budding of biological membranes IGLIČ 1, A., URBANIJA 3, J., HAGERSTRAND 2, H., AND KRALJ-IGLIČ 1,3, V. Laboratory of Physics, Faculty of Electrical Engineering, University of Ljubljana, Trzaska 25, SI-1000 Ljubljana, Slovenia Department of Biology, Åbo Akademi University, Biocity, FIN-20520 Åbo/Turku, Finland 3 Laboratory of Clinical Biophysics, Faculty of Medicine, University of Ljubljana, Lipiceva 2, SI-1000 Ljubljana, Slovenia 1 2

It is shown that tubular budding of biological membrane may be explained by in-plane ordering of anisotropic membrane nanodomains in the buds where the difference between the principal membrane curvatures is very large. In contrast to previously reported theories, no direct external mechanical force is needed to explain tubular budding of the bilayer membrane. We also show that spherical budding of the biological membrane is driven by a local increase of the area difference between the outer and inner lipid layer, by accumulation of anisotropic molecules or raft elements in the necks connecting the bud and the parent membrane and by accumulation of isotropic molecules or membrane nanodomains on the bud. It is also shown that the attachment of proteins to the membrane surface may cause the coalescence of the membrane bud (spherical daughter vesicle) and the parent membrane. Theoretically, the protein-mediated attraction between the membranes was described as an interaction between two electrical double layers. It was found that a specific spatial distribution of the charge within the proteins attached to the membrane surface may explain the observed attraction.

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LECTURE ABSTRACTS Wednesday 22nd August

11:30-12:00

Parallel Session b.1.1. Connection between the function of hydrogenase enzyme and photosynthetic electron transport in Thiocapsa roseopersicina BBS NAGY 1, L., NOVAK 2, J., DOROGI 1, M., LATINOVICS 2, D., KOVÁCS 2,3 K.L. AND RÁKHELY 2,3, G. Department of Medical Physics and Biophysics, University of Szeged, Egyetem str. 2 Szeged, H-6726, Hungary Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences Temesvari str. 62., Szeged, H-6726, Hungary 3 Department of Biotechnology, Unversity of Szeged, Temesvari str. 62., Szeged, H-6726, Hungary, [email protected] 1 2

Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium perfoms anaerobic photosynthesis using reduced sulfur compounds as electron supply. The strain contains at least one soluble (Hox) and two membrane-associated (Hyn, Hup) [NiF] hydrogenases [1,2]. It was recently demonstrated, that the cells, grown in the presence elevated thiosulfate in the medium, can evolve photobiohydrogen [3]. Inversely, in dark, low thiosulfate concentration stimulates the hydrogen production. In both cases, the socalled NAD+-reducing Hox hydrogenase is responsible for the hydrogen evolution. The HoxYH subunit of this hydrogenase is responisble for the proton reduction/hydrogen oxidation, while the hoxFU genes encode for the diaphorase subunits. The fifth subunit, the HoxE, is required for the in vivo but not for the in vitro hydrogenase activity, therefore, this subunit likely has an electron transferring role. This means a third electron input-output domain for this protein complex and the Hox hydrogenase is suggested to function as an electron valve between the various processes. Moreover, the in vivo hydrogen evolving capacity could be blocked by using Nuo inhibitors and significantly reduced by photosynthesis inhibitors, hence, it seems that the virtually cytoplasmic Hox hydrogenase has direct connection to the respiratory chain and photosynthesis. The electron transport within and around the photosynthetic reaction centers (RC) was characterized in the intracytoplasmic membranes (ICMs) isolated from wild type (BBS) and HoxE mutant Thiocapsa roseopersicina BBS. ICMs were suspended in 10 mM TRIS, 100 mM NaCl (pH:8.0) and the kinetics of flash induced absorption change was measured at 860 nm. 10 µM K3[Fe(CN)6], 400 µM terbutryne (TERB), 2 µg/ml antimycine (ANT) was added, if necessary. At this wavelength, the redox state of the primary donor (P/P+) can be followed. The measured decay curves were decomposed into three components by multiexponential analysis. The slowest component (τ ≈ 10 s, A(%) ≈25-35) corresponds to the redox equilibrium on the acceptor side through the secondary quinone, QB. The medium one (τ ≈ 0.1 s, A(%) ≈ 4050) corresponds to the P+QA- → PQA charge recombination, while the faster one (τ ≈ 10-50 ms, A(%) ≈30) reflects on the electron equilibrium on the donor side through cytochrome. The addition of TERB did not block the charge recombination from the QB-site (39% in the presence of TERB and 17% for the TERB+ANT sample) even in this high concentration, which indicates that after dark adaptation there is a considerable part of RCs in the reduced QB- state. The contribution of the slow component is even higher (75%) in the presence of TERB and (57%) for TERB+ANT treated membranes, which indicates that the electron equilibrium in the membrane is shifted to the direction of the RCs from the hydrogenase in the mutant, which further corroborates the interrelationship of the photosynthesis and the hydrogen metabolism. Acknowledgement This work has been partly supported by EU 6th Framework Programme projects (HyVolution SES6 019825 and NEST STRP SOLAR-H 5166510), and by domestic funds (NKTH, GVOP, Asbóth, Baross, DEAK-KKK, KN-RET) References [1] Rákhely, G, Kovács ÁT, Maróti G, Fodor B, Csanádi G, Latinovics D and Kovács KL (2004) Appl. Environ. Microbiol. 70:722-8 [2] Kovács, KL, Maróti, G and Rákhely G (2006) International Journal of Hydrogen Energy 31:1460 – 1468 [3] Rákhely G, Laurinavichene TV, Tsygankov AA, Kovács KL (2007) Biochim Biophys Acta DOI 10.1016/j.bbabio.2007.02.004

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LECTURE ABTRACTS nd

Wednesday 22 August

12:00-12:30

Parallel Session b.1.2. Protein relaxation: case-history of bacterial reaction center MARÓTI 1, P., FILUS 1, Z., SEBBAN 2, P., WRAIGHT 3, C.A. AND LACZKÓ 1, G. Department of Biophysics, University of Szeged, Egyetem utca 2, Szeged, H-6722, Hungary, [email protected] Laboratoire de Chimie Physique, CNRS, Faculté d’Orsay, Orsay/UMR 8000, University of Paris XI, 91405, France 3 Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 505 S. Goodwin Av, Urbana IL, USA 1 2

Efficient transduction of light energy into biochemical energy occurs in membrane proteins of plants and bacteria during photosynthesis. The best characterized photosynthetic system is the reaction center (RC) protein from purple non-sulfur bacteria, with serves as an obvious and effective model system for the photosystems of higher plants. However, it has also been established as the premier system for studying biological electron transfer, and mechanisms of dynamic charge compensation in proteins. Considering that 1/3 of all known proteins are redox active and almost all known enzymatic mechanisms involve proton transfer, these are issues of major significance for understanding protein function generally. This presentation will utilize the unique advantages of the bacterial system: knowledge of the RC’s three dimensional structure, well established methods for modifying the protein and the cofactors, the many distinct reaction steps covering a broad range of times and energies, and the diversity of spectroscopic tools for following the progress of events. Excitation of the RC by a short flash of light induces charge separation followed by a series of electron transfer reactions. The resulting electrostatic perturbations drive the protein to relax over a very wide (from picosecond to second) time scale. The protein relaxation includes various structural responses such as conformational changes, charge redistributions and proton uptake by the RC, with functional consequences including conformational gating of electron transfer. The goal of the talk is to understand the role of protein dynamics in determining the kinetics and thermodynamics of biological catalysis, specifically electron and proton transfer reactions involving the primary (QA) and secondary (QB) quinones of RCs of Rhodobacter (Rb.) sphaeroides We will emphasize the use of delayed fluorescence (arising from back reactions generating the excited state of the primary donor, P*) as a quantitative probe of the time-dependent energetics of the charge separated states [1,2]. Comparison with spectroscopic measurements will reveal the time course of electronically silent states (protein relaxation) that contribute to the reaction dynamics. By using electronic gating of the photodetector, we measured delayed fluorescence in the microsecond time range - a time domain that has not previously been explored [3]. This will allow us to study the relaxation behavior of P+QA- and P+QB- on this time scale and longer. More specific identifications will be made by using native and artificial quinones and site-directed mutants to affect the energetics of the quinone states, and to identify the protein-cofactor interactions that constitute the in situ solvation of the transient states. Acknowledgement Thanks are due to OTKA, NSF and Balaton (TéT) for the financial support. References [1] Turzó, K, Laczkó, G, Filus, Z, Maróti, P (2000) Biophysical Journal 79: 14-25. [2] Nagy, L, Milano, F, Dorogi, M, Agostiano, A, Laczkó, G, Szebényi, K, Váró,Gy, Trotta, M, Maróti, P (2004) Biochemistry 43, 12913-12923. [3] Filus, Z., Laczkó, G., Wraight, C.A., Maróti, P (2004) Biopolymers, 74 (1-2), 92-95.

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LECTURE ABSTRACTS Wednesday 22nd August

12:30-13:00

Parallel Session b.1.3. Light-induced ultrafast electron and proton motion in bacteriorhodopsin generates THz radiation GROMA 1, G.I., HEBLING 2, J., KOZMA 3, I.Z., VÁRÓ 1, G., KUHL 4, J. AND RIEDLE 3, E. Institute of Biophysics, Biological Research Center, H-6726 Szeged, Hungary, [email protected] Department of Experimental Physics, University of Pécs, H-7624 Pécs, Hungary 3 Chair for BioMolecular Optics, Ludwig-Maximilians-University, D-80538 Munich, Germany 3 Max Planck Institute for Solid State Research, D-70569 Stuttgart, Germany 1 2

Previous electric measurements on oriented purple membranes of bacteriorhodopsin (bR) carried out in the picosecond to second time range revealed the strict correlation between the photocycle and the step-bystep transport of protons across the purple membrane. On the other hand, the primary electrogenic events taking place during and right after the excited state of the retinal chromophore have remained unexplored. This is mainly due to the technical limitations of the previous experimental procedures. Based on the detection of electromagnetic radiation generated by ultrafast intramolecular charge translocation, here we report an alternative approach for monitoring the above processes. In this method the emissions from the individual molecular antennae are summed up forming a detectable signal, due to their microscopic ordering in the purple membrane and the possibility of macroscopic orientation of the whole sample. Our results represent the first time-resolved observation of THz radiation from a biological object. The time domain THz emission from dried oriented purple membranes excited by 100 fs laser pulses was detected in the 0.1 – 3 THz range by electrooptic sampling. Experimental arrangements for far- as well as near-field observation resulted in a high weight of the faster and the slower components of the radiation, respectively. The detected signal, spanned in an ~8 ps time window, could be clearly decomposed into a dominating fast and a small but definite slow phase. Several theoretical models were tested to describe the kinetics of the radiation from both native bR as well as its acid blue form, having prolonged excited state life-time. In a numerical simulation procedure, we followed the time evolution of the radiated electric field from creation to detection. Our results suggest that the major component of the signal is originated from an intramolecular electron transfer process occurring in the excited state of the retinal. The slower phase has an opposite amplitude and correlates well with the formation of the K intermediate of the photocycle. It is attributed to a primary step of the proton pump, presumably taking place in an H-bond around the retinal. The harmonized occurrence of the ultrafast electron and proton translocation supports the idea assigning important functional role to the light-induced electron polarization in the energy conversion mechanism of bR. From the point of view of Mitchell’s chemiosmotic theory, this corresponds to the extension of it into the territory of ultrafast electrodynamics.

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LECTURE ABTRACTS nd

Wednesday 22 August

14:00-14:20

Company Presentation 1. Raman microscopy: A powerful tool for probing biological systems ZOUBIR 1, A., MOREL 1, S. AND FITZGERALD 1, S. 1

HORIBA JOBIN-YVON SAS, 231 rue de Lille, 59650 Villeneuve d’Ascq, France, [email protected]

Raman spectroscopy is a fast-developing technique for probing cellular biochemistry at the micron scale. In contrast to other imaging techniques, Raman microscopy provides images where each pixel contains full spectral information. Coupled to a standard upright microscope or to an inverted microscope, this technique enables confocal images with high spatial resolution. Such images can be used for studying the chemistry of individual cells or particles, and for measuring the components distribution of complex biological systems. Several applications related to biophysics will be described. For instance, fingerprint spectra can be obtained from single bacteria cells. Tentative assignment of the main bands shows contribution from nucleic acids, carbohydrates, proteins and lipids, from which different bacteria species can be distinguished through chemometric analysis. Further studies have shown that it is also possible to distinguish growth stage, and to identify quantify uptake of isotope labelled nutrients. Surface-Enhanced Raman Scattering (SERS) using colloidal gold solutions enables species present in extremely low concentrations to be identified within seconds. This technique has been used to investigate intracellular localisation of chemotherapeutic drugs in single living lymphocytes, intracellular interactions of anti-cancer drug mitoxantrone, and disease diagnostic of tissue. Fig. 3 shows a Raman image of healthy colonic tissue illustrating how the presence of various chemicals can be identified within the structural features of the tissue. Comparison between data obtained for healthy and diseased tissue enables scientists to not only learn more on the biochemical changes caused by the cancer, but also to categorise tissue according to disease state.

Fig. 1. Analysis results for human colonic tissue section (A) optical image of histopathologic HE stained tissue, and (B) Raman mapped image illustrating intensity of the Amide I band.

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LECTURE ABSTRACTS Wednesday 22nd August

14:20-14:40

Company Presentation 2. Broadband and Broadly Tunable Femtosecond Pulse Laser and Parametric Oscillators for Applications in Femtobiology R. SZIPİCS 1, A. MAKAI 1,2, P. SÁNDOR 1, J. HEBLING 1 AND G. GROMA 2 1 2

R&D Ultrafast Lasers Ltd, Attila út 73, Budapest, H-1012, Hungary, [email protected] Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62, H-6701, Szeged, Hungary

Broadband and broadly tunable femtosecond pulse laser and parametric oscillators play an important role in several biological and medical applications such as time resolved-spectroscopy, nonlinear microscopy or other types of imaging techniques such as fluorescence lifetime imaging (FLIM). The bandwidth and the tuning range of femtosecond pulse oscillators were considerably extended by the invention of ultrabroadband chirped mirrors [1]. The mirrors exhibit high reflectivity (R > 99.6%) and smooth variation of group delay versus frequency over several hundreds of nanometers. In case of Ti:sapphire lasers, this feature allows mode-locked operation over the whole tuning range (670 nm -1040 nm) using one set of ultrabroadband chirped mirrors only. This makes them attractive for two-photon absorption fluorescence imaging applications in which several fluorophores with highly separated absorption bands should be used [2]. The tuning range of Ti:sapphire lasers can be further extended by nonlinear frequency conversion techniques such as second-harmonic generation (SHG) and optical parametric wave generation. For this purpose, we developed synchronously pumped optical parametric oscillators (OPOs) [3], in which periodically poled lithium niobate (PPLN) crystals are used as a nonlinear medium for frequency conversion. Low coherence light sources are important tools in biomedical imaging. Taking the advantage of our patented chirped mirror techology [4] we have developed ultrashort pulse (τ < 10 fs) laser oscillators exhibiting very broad spectrum (∆λ > 80 nm), i.e., low coherence length, hence they are well suited for optical coherence tomography (OCT). Further details on our femtosecond pulse lasers can be found in [5]. Spectrometer

Ti:S pump laser (λ = 710 - 880 nm; ∆τ = 100 fs)

HR

SHG Ti:S

Piezo control

PC

HR DM

HR

HR

CM

CM

CM

CM

CM

CM

HR HR

SHG OPO

HR HR

HR

P

BS HR Filter

Fiber

HR HR

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PPLN DM

DM

Delay

For femtosecond pulse time-resolved spectroscopic studies, we have developed a pumpprobe experimental setup comprising our FemtoRose 100 TUN broadly tunable Ti:sapphire laser and our FemtoRainbow IR OPO (see Figure 1) [5]. Their outputs are frequencydoubled, which results in tunable excitation and probe pulses over the 355-440 nm and 520-650 nm wavelength regimes, respectively.

HR

Output:

SHG TI:S (λ = 355 - 440 nm; ∆τ = 100 fs) SHG OPO (λ = 520 - 650 nm; ∆τ = 100 fs)

Legends: DM – dichroic mirror; L – lens; Fiber – multimode optical fiber; CM – chirped mirrors; P – piezo translator; PPLN – periodically poled lithium niobate; HR – high reflector; Filter – color filter; SHG – second-harmonic generation; Delay – delay line; BS – beam splitter

Figure 1. Femtosecond pump-probe setup for time resolved transient absorp-tion measurements.

References [1] Mayer EJ, Möbius J, Euteneuer A, Rühle WW, Szipıcs R (1997) Opt. Lett. 22: 528-530. [2] Rozsa B, Katona G, Vizi ES, Várallyay Z, Sághy A, Valenta L, Maák P, Fekete J, Bányász Á, Szipıcs R (2007) Appl. Opt. 46: 1860-1865. [3] Hebling J, Mayer EJ, Kuhl J, Szipıcs R (1995) Opt. Lett. 20: 919-921. [4] U.S. Pat. No. 5,734,503 [5] www.fslasers.com

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LECTURE ABTRACTS nd

Wednesday 22 August

14:40-15:00

Company Presentation 3. Advances in Lasers for Multiphoton Excitation Microscopy PAUL REINHARD Coherent GmbH., [email protected]

Multiphoton Excitation (MPE) microscopy had become an important bioimaging technique, enabling the study of dynamic processes in living cells and tissues without causing significant damage. MPE produces high-resolution, three-dimensional images, and primarily relies on the use a tunable, ultrafast laser to excite highly specific fluorophores in order to follow specific biochemical processes. Key to future advances in MPE is the ability to work with a wider range of fluorophores, to increase data acquisition speed, and to improve data signal-to-noise ratio. In terms of laser characteristics, these requirements translate into wider tuning range, faster tuning speeds, and higher peak power delivered to the sample. This paper explores the advances in tunable ultrafast laser and amplifier technology currently under development to meet these goals and thus power the next generation of MPE instrumentation.

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LECTURE ABSTRACTS THURSDAY, 23RD AUGUST Thursday 23rd August

8:30-9:05

Plenary Lecture 3.1. Combating Amyotrophic Lateral Sclerosis with Biophysical Means ANDJUS , P.R. Dept. of Physiol. and Biochem., Sch. of Biol., Univ. of Belgrade, Studentski trg 3; 11000 Belgrade, Serbia, [email protected]

Amyotrophic lateral sclerosis (ALS) a devastating neurological disorder affecting upper and lower motoneurons. It is characterized by progressive muscle weakness and atrophy. It is almost invariably fatal, usually within 3 to 6 years after the beginning of the symptoms. There are two types, sporadic and familial that differ in the aetiopathogenesis although they have similar clinical symptoms and neuropathology. Moreover, there is substantial evidence that both types of ALS develop an oxidative stress phase (by extensive glutamate stimulated Ca2+ inflow or by impaired SOD activity) that eventually leads to excitotoxicity. The cause of sporadic ALS is not known but several theories have been proposed, including a) excitotoxic stimulation due to accumulation of glutamate, and b) immune mechanisms. Pathogenic Igs from ALS patients were suggested as the link between the immune mechanisms and the excitotoxicity hypothesis. Neuroimmunological study of ALS was performed on hippocampal neurons in culture. Sera from sporadic ALS patients were used to isolate IgGs. Direct ALS IgG effect on Cai2+ homeostasis was measured with confocal microscopy with a fluorescent dye selective for Ca2+ (fluo-3). ALS IgGs specifically blocked, in an irreversible manner, the P/Q type channel activity. An excitotoxic hypothesis was still applicable assuming a suppression of the Cai2+ -dependent desensitization of NMDA receptors. ALS IgGs also induced a delayed Cai2+ transient response to KCl or glutamate in glial-like cells. ALS IgG fraction can indeed affect the presynaptic glutamate release mechanisms as shown in a whole-cell patch clamp study of spontaneous non-NMDA excitatory postsynaptic currents (SEPSC). ALS IgGs focally applied above the cell induced a 3fold rise in frequency of SEPSC and a 2-fold rise in frequency of miniature EPSCs (in TTX). No changes were observed with IgGs from healthy donors or from patients with Alzheimer’s disease. Neither amplitude nor decay time of the EPSCs were affected by ALS IgGs, which also did not modify the amplitude or shape of currents evoked by the non-NMDA agonist, AMPA - all pointing to a presynaptic site of action. Recent results on the model of familial ALS, the transgenic hSOD1 G93A mutant rat demonstrated its feasibity for an in vivo follow-up study. By means of a wide bore 1.5 T MRI instrument and a surface head coil we managed to image degenerative foci (hyperintensity changes) in the brain and brainstem of the model rat. The hyperintensity changes could be observed already in presymptomatic animals but became more apparent in later stages of the disease. Immunocytochemistry of these regions revealed reactive astroglia and by means of SOD1 antibodies and ED1 marker of activated microglia/macrophages revealed a neuron-glia interaction. In addition, MRI scanning with a 3 T wide bore magnet revealed infiltrations of magnetically labeled T cells in the brainstem. There is still considerable work to be done on the relationship of two forms of ALS, however our studies point that the common denominator may be the function of glial cells. Acknowledgement This work was supported by MSEP grant #143054B to A.P.R.

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LECTURE ABTRACTS rd

Thursday 23 August

9:05-9:40

Plenary Lecture 3.2. Water transport through membrane channels POHL, P. Institut für Biophysik, Johannes Kepler Universität , Linz, 4040, Austria

Confinement of water by pore geometry alters its physical characteristics. Changes in flow dynamics are partially due (i) to the reduction in number of hydrogen bonds compared to bulk fluid, and (ii) due to interactions of the permeating molecules with the channel wall. With decreasing channel diameter and increasing hydrophobicity the probability of liquid vapour oscillations increases [1]. The resulting intermittency of pore conductance supports the theory of hydrophobic gating. This theory explains how transmembrane pores that are incompletely occluded in their closed state prevent the movement of solutes and solvent molecules. It makes the following testable predictions: (i) The velocity of transport across the channel may increase beyond the diffusion limit: (ii) The density of water molecules in the channel is lower than in bulk; (iii) The probable presence of vapour in the pore would influence the relative stability of NH3 and NH4+ in the pore. Experimental evidence compatible with all three predictions will be presented. It was obtained in studies of water permeability of purified and reconstituted membrane channels. An osmotic gradient was imposed across reconstituted planar lipid bilayers and the resulting shift in solute concentration close to the membrane surface was detected by scanning microelectrodes. It allowed calculation of the single channel permeability coefficient pf and water mobility inside the channel. Peptidic nanopores, ion channels and aquaporins were investigated. The bacterial potassium channel KcsA exhibited the highest pf. Water mobility inside the potassium channel exceeded bulk mobility 20-fold [2]. In contrast, water mobility inside short peptidic nanopores was equal to bulk mobility. It decreased exponentially with increasing pore length. An increment of just one accommodation site for water resulted in a fourfold drop of pf [3]. The non-linear dependence of pf on channel length indicated that several accommodation sites for water were unoccupied [3]. In view of the vapour-like density of water, we tested whether water conducting channels may provide a transport pathway for NH3 as well. In agreement with the hypothesis, the water channel aquaporin-8 was found to transport neutral ammonia molecules as fast as it does convey water molecules [4]. After having confirmed the predictions (i – iii), we checked whether the hydrophobic gating theory is applicable to the bacterial peptide translocation channel SecY. A point mutation in the hydrophobic pore ring, a narrow constriction zone in the center of the channel confirmed that the ring is required to seal the channel in its closed state. However, the pore ring alone did not prevent the passage of ions over periods of seconds or minutes, as shown by our electrophysiology experiments [5]. At least in the case of SecY hydrophobic gating has to be accompanied by additional gating mechanisms to maintain the membrane barrier. Acknowledgement Financial support of the Austrian Science Fund is acknowledged. References [1] Beckstein O, Sansom MSP (2003) Proc. Natl. Acad. Sci. U. S. A. 100: 7063-7068. [2] Saparov SM, Pohl P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101: 4805-4809. [3] Saparov SM, Pfeifer JR, Al-Momani L, Portella G, de Groot BL, Koert U, Pohl P (2006) Phys. Rev. Lett. 96, 148101. [4] Saparov SM, Liu K, Agre P, Pohl P (2007) J. Biol. Chem. 282:5296-5301. [5] Saparov SM, Erlandson K, Cannon K, Schaletzky J, Schulman S, Rapoport TA, Pohl P (2007) Mol. Cell in press.

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LECTURE ABSTRACTS Thursday 23rd August

9:40-10:15

Plenary Lecture 3.3. Coupling of activation and inactivation gating of Shaker K+ channels PANYI 1, G., AND DEUTSCH 2, C. 1 2

University of Debrecen, Dept. of Biophysics and Cell Biology, 98. Nagyerdei krt., Debrecen, 4012, Hungary, [email protected] University of Pennsylvania, Dept. of Physiology, 3700 Hamilton Walk, Philadlephia, PA, 19104-6085, USA.

Voltage-gated potassium (Kv) channels in the Shaker subfamily have three well-studied gates, an activation gate and two types of inactivation gates. The gate responsible for fast (N-type) inactivation is well established, whereas the molecular mechanism of slow (P/C-type) inactivation is less well understood but is known to involve a rearrangement of the selectivity filter (P-gate). Although the movements of these individual gates have been studied extensively in isolation, the energetic and kinetic coupling of these gates has not been demonstrated directly. We have addressed the issue of energetic coupling between the activation and slow inactivation gates by monitoring the status of the activation gate in slow inactivated Shaker-IR (N-type inactivation deficient) channels. Because inactivated channels are nonconducting, we designed two novel protocols to assess the status of the activation gate. The kinetics of closing of the activation gate were measured by time-dependent changes in the kinetics of the accessibility of a cysteine residue engineered into the lining of the pore cavity (V474C). Opening of the activation gate in slow inactivated channels was monitored by the kinetics of the liberation of a Cs+ ion trapped behind the closed activation gate. Our study clearly indicates that the activation and inactivation gates of Shaker channels are coupled: a closed inactivation gate favors faster opening and slower closing of the activation gate. We also showed that hyperpolarization closes the activation gate long before a channel recovers from inactivation, thus, the ratelimiting step in recovery from inactivation is not the closure of the activation gate. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the crosstalk between them is likely to be a fundamental factor in controlling ion flux across membranes. What molecular mechanism underlies coupling? Although slow inactivation involves a local rearrangement of the outer mouth of Kv channels, rearrangements may also occur in the cavity between the activation gate and the selectivity filter. To test this hypothesis, we measured the kinetics of modification of strategically positioned cysteine residues (V474C and I470C) by different cysteine reagents (MTSET, MTSEA, Cd2+). The accessibility of residues facing the aqueous cavity is dramatically different in open vs. inactivated channels. Furthermore, inactivation of the channels is accompanied by a ~16-fold reduction in the affinity of a blocker, tetraethylammonium, for its internal binding site as compared to the open state. We conclude that the cavity of the slow-inactivated Kv channel is conformationally different from that of

the open channel and that a propagated conformational change in the cavity is responsible for coupling between activation and slow-inactivation gates. Acknowledgement Supported by National Institutes of Health Grant GM 069837 and NS 052665, Hungarian Ministry of Health ETT 068/2006 and 064/2006 and Hungarian National Research Fund OTKA K 60740 and NK 61412. Gyorgy Panyi is a Bolyai Fellow.

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LECTURE ABTRACTS rd

Thursday 23 August

10:15-10:50

Plenary Lecture 3.4. Spin Label EPR Spectroscopy of the Vacuolar Proton-ATPase: Structure, Function and Inhibitors PÁLI 1,2, T. AND MARSH 1, D. 1 2

Abteilung Spektroskopie, Max-Planck-Institut für biohysikalische Chemie, Fassberg 11, Göttingen, 37077, Germany Institute of Biophysics, Biological Research Centre, Temesvári krt. 62, Szeged, 6726, Hungary

The vacuolar proton-ATPase (V-ATPase) is a membranous molecular motor, which uses energy from ATP hydrolysis to drive trans-membrane proton transfer. It works in the opposite sense as the better known F-ATPase. Our long-term objective is to reveal the mechanism of this enzyme and to identify specific inhibitor binding sites/conditions that would have a direct medical relevance. Biomembranes were purified from lobster, containing a 16-kDa protein of the ductin family [1] that is capable of functionally substituting subunit c of the yeast V-ATPase in a hybrid system, or from yeast. Conventional and non-linear electron paramagnetic resonance (EPR) techniques were used to gain structural data on the intramembranous subunit c of V-ATPase. Spin labelled lipids were used to determine the oligomeric state of the proton channel protein by detecting the first shell of lipids interacting with its transmembrane region [2]. Structurally and functionally important unique cystein and glutamate residues were spin-labelled for their localisation along the membrane normal. Spin label - spin label interaction and paramagnetic quenching studies also revealed the oligomeric state to be a hexameric, and the unique cystein and glutamate residues have been shown to at least partially contact lipids [3]. A divalent-ion binding site on the proton channel has been revealed by detecting endogenous Cu2+ and by EDTA washing followed by Ni2+ binding experiments [4]. Perturbation of both the spin labelled amino acid side chains and spin labelled annular lipids around the proton channel by hydrophobic inhibitors of V-ATPase has been demonstrated for the yeast and lobster proteins [5-7]. Our data set constraints on future structural models and aid inhibitor design. Acknowledgement Supported in part by the European Commission (QLG-CT-2000-01801), the Volkswagen-Stiftung and the Hungarian National Science Fund (OTKA T029458, T043425 and K68804). References [1] Holzenburg, A, Jones, PC, Franklin, T, Pali, T, Heimburg, T, Marsh, D, Findlay, JBC and Finbow, ME (1993) European Journal of Biochemistry 213(1): 21-30. [2] Pali, T, Finbow, ME, Holzenburg, A, Findlay, JBC and Marsh, D (1995) Biochemistry 34, 9211-9218. [3] Pali, T, Finbow, ME and Marsh, D (1999) Biochemistry 38(43): 14311-14319. [4] Pali, T, Finbow, ME and Marsh, D (2006) Biochimica et Biophysica Acta - Biomembranes 1758: 206-212. [5] Pali, T, Dixon, N, Kee, TP and Marsh, D (2004) Biochimica et Biophyisica Acta - Biomembranes 1663: 14-18. [6] Dixon, N, Pali, T, Kee, TP and Marsh, D (2004) Biochimica et Biophyisica Acta - Biomembranes 1665: 177-183. [7] Pali, T, Whyteside, G, Dixon, N, Kee, TP, Ball, S, Harrison, MA, Findlay, JBC, Finbow, M and Marsh, D (2004) Biochemistry 43: 12297-12305.

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LECTURE ABSTRACTS Thursday 23rd August

11:30-12:00

Parallel Session a.2.1. pH-dependent regulation of the inactivation of a lymphocyte potassium channel VARGA, Z., SOMODI, S., HAJDU, P., GÁSPÁR, R., PANYI, G. University of Debrecen, Dept. of Biophysics and Cell Biology, Nagyerdei krt. 98., Debrecen, 4012 Hungary, [email protected]

The voltage-gated Kv1.3 potassium channel plays an essential role in the control of the membrane potential and antigen-stimulated proliferation of human lymphocytes. It has been shown that pharmacological manipulation of these channels can modulate certain immune processes making them potential drug targets in the future. It is therefore crucial to know the details of the gating mechanisms of Kv1.3 and the factors, such as extracellular pH and potassium concentration that regulate gating. During periods of prolonged depolarization the Kv1.3 channel enters a non-conducting inactivated state, and this process occurs exclusively via the slow C/P-type mechanism that means a conformational change in the selectivity filter of the channel. This process is controlled by a “foot-in-the-door” mechanism, which means that a K+ ion binding site must be vacant in order for inactivation to proceed. While acidification of the extracellular solution accelerates the rate of slow inactivation of all related potassium channels, the opposite, that is, acceleration is observed for Kv1.3 channels. There is a titratable histidine residue in a critical position of the pore region (H399, equivalent of Shaker 449), which was previously shown to influence the rate of slow inactivation. With our experiments we investigated the role of this histidine in the anomalous pH-dependence of the inactivation of Kv1.3. We suggested a model in which the electric field of the histidines protonated at low pH creates a potential barrier for K+ ions just outside the external mouth of the pore that hinders K+ ion movement between the extracellular medium and the binding site controlling inactivation. Thus, depending on the conditions that determine the direction from which the site is mostly filled by K+ ions, the rate of inactivation may be accelerated or decelerated. We tested the validity of our model with different approaches. First, we used a high ionic strength solution because this reduced the range of electrostatic interactions and the slowing effect of the protonated histidines on the rate of inactivation was inhibited. Second, we showed that barium ions that have a very similar size to K+ ions, but block the channel, enter and exit the channel pore more slowly at low pHext. Third, if the current direction was reversed the slowing of inactivation by low pHext could not be observed. Our results with mutant channels, in which H399 was changed for a permanently neutral or positively charged residue provided further proof for our model. Thus, we have successfully described the mechanism by which pHext modifies the rate of inactivation of Kv1.3. The pH-dependence of slow inactivation may be an important regulatory mechanism, for example at areas of inflammation, where the local pH may deviate from the physiological value significantly. Acknowledgement This work was supported by grants ETT 076/2006 to Z.V, ETT 068/2006 and OTKA K 60740 to G.P. and ETT 064/2006 to R.G.

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LECTURE ABTRACTS rd

Thursday 23 August

12:00-12:30

Parallel Session a.2.2. Sulfonylurea Receptors Type 1 and 2A Readily Coassemble to Form Octameric KATP Channels of Mixed Subunit Composition CSANÁDY 1, L. AND CHAN 2, K.W. 1 2

Semmelweis University, Dept.Med. Biochem., Puskin u. 9, Budapest, 1088, Hungary, [email protected] Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4970, USA

ATP-sensitive potassium (KATP) channels play important roles in regulating insulin secretion, cardiac contractility and vascular tone. KATP channels are heterooctamers of four pore-forming inward rectifier (Kir6.2) subunits and four sulphonylurea receptor (SUR) subunits. KATP channels containing SUR isoforms 1 (pancreatic) and 2A (cardiac) display distinct gating characteristics and pharmacological profiles. Expression of both SUR1 and SUR2 together with Kir6.2 has been reported in some cell types raising the possibility that mixed channels containing both types of SUR subunits might exist. To test whether SUR1 can coassemble with SUR2A to form functional KATP channels we constructed tandem constructs from an SUR fused to either a wild-type (WT) or a mutant N160D Kir6.2 subunit. The latter mutation greatly increases the sensitivity of KATP channels to block by intracellular spermine. We expressed, individually and in combinations, tandem constructs SUR1-Kir6.2 (S1-WT), SUR1-Kir6.2(N160D) (S1-ND), and SUR2AKir6.2(N160D) (S2-ND) in Xenopus oocytes, and studied the voltage-dependence of spermine block in inside-out macropatches over a range of spermine concentrations and RNA mixing ratios. Either tandem construct alone supported macroscopic ATP-sensitive K+ currents, with low spermine sensitivity in the case S1-WT, and high spermine sensitivity in the case of S1-ND and S2-ND. Coexpression of S1-WT with S1ND caused the appearance of current components with intermediate spermine sensitivities indicating the presence of populations of channels containing both types of Kir subunits at all possible stoichiometries. The relative abundance of these populations, determined by global fitting over a range of conditions, followed binomial statistics suggesting that WT and N160D Kir6.2 subunits coassemble indiscriminately. Coexpression of S1-WT with S2-ND also yielded current components with intermediate spermine sensitivities suggesting that SUR1 and SUR2A readily coassemble into functional KATP channels. Slight deviation from binomial statistics of the distribution of these populations points to only small differences in interaction energies between SUR1/SUR1, SUR1/SUR2A, and SUR2A/SUR2A. Acknowledgement Funded by NIH grant DK60104 (to K.W. Chan). L. Csanády is a Bolyai Research Fellow of the Hungarian Academy of Sciences.

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LECTURE ABSTRACTS Thursday 23rd August

12:30-13:00

Parallel Session a.2.3. Intracellular Ca2+ signalling in hepatocytes after inhibition of plasma membrane Ca2+ efflux GOSAK 1, M., GREEN 2, A. K., DIXON 3, C.J. AND MARHL 1, M Dept. of Physics, Faculty of Natural Sciences and Mathematics, University of Maribor, Koroška c. 160, Maribor, SI-2000, Slovenia, [email protected] 2 Dept. of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK 3 Leicester School of Pharmacy, Cell Signalling Lab, De Montfort University, The Hawthorn Building, Leicester, LE1 9BH, UK 1

Many non-excitable eukaryotic cell types, including hepatocytes, respond to extracellular agonists, such as hormones or neurotransmitters, by generating oscillatory changes in concentration of free cytosolic Ca2+ (Ca2+ oscillations). Ca2+ oscillations play a vital role in intra- and intercellular signaling. Many cellular processes, such as secretion or egg fertilization are regulated by Ca2+ oscillations. We have shown experimentally that glucagon-(19-29) (mini-glucagon) and carboxyeosin inhibit Ca2+ efflux from populations of intact rat hepatocytes [1]. In further experiments we have shown that the carboxyeosin and mini-glucagon enhance the frequency of Ca2+ oscillations induced by Ca2+-mobilizing agonists in single hepatocytes, but do not affect the duration of individual transients. The enhanced Ca2+ oscillation frequency resulting from Ca2+ efflux inhibition can be explained by the previous mathematical models for Ca2+ oscillations in non-excitable cells (for review of the models see [2,3]). However, none of the models is able to explain the experimental that while the frequency of Ca2+ oscillations is increased, the duration of individual transients is not affected. Here we show that the problem can be solved by partitioning the cell into sub-compartments. The dynamics of every particular part is governed by a complete set of model equations [4] and the Ca2+ can diffuse between the individual sub-compartments. The experimental conditions of inhibited Ca2+ efflux (and allowed Ca2+ influx into the cell) are simulated by introducing a varying Ca2+ flux into the exterior 10% of the cell, which resembles the fact that the influx cannot affect the whole cell volume. Since the calcium concentration is measured on the basis of the luminescence of the entire cell, in the model it is sufficient to consider the mean-field dynamics of all cellular compartments. Indeed, when the mean-field signal is considered, an increase in spiking frequency is observed, whereby the width of the spikes remains constant. We therefore show that due to the spatial extension of the cell model our theoretical results are in much better agreement with the experimental observations than those predicted by previous non-spatial extended mono-compartmental models. Acknowledgement

A grant from British Council and Slovenian Research Agency (BI-GB/06-013) is gratefully acknowledged. Anne K. Green (grant 065845) and C. Jane Dixon (grant 059089) are also grateful to the Wellcome Trust for funding. References [1] Green, A.K., P.H. Cobbold, C.J. Dixon. Effects of the hepatocyte [Ca2+]i oscillator of inhibition of the plasma membrane Ca2+ pump by carboxyeosin or glucagon-(19-29). Cell Calcium 22 (1997) 99-109. [2] Schuster, S., M. Marhl, and T. Höfer. Modelling of simple and complex calcium oscillations. From single-cell responses to intercellular signaling, Eur. J. Biochem. 269 (2002)1333-1355. [3] Falcke, M. Reading the patterns in living cells - the physics of Ca2+ signaling. Adv. Phys. 53 (2004) 255-440. [4] Marhl, M., T. Haberichter, M. Brumen, R. Heinrich. Complex calcium oscillations and the role of mitochondria and cytosolic proteins. BioSystems 57 (2000) 75-86.

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LECTURE ABTRACTS rd

Thursday 23 August

11:30-12:00

Parallel Session b.2.1. Elasticity and Breakdown of Soft Biological Tissues SUKI 1, B., JESUDASON, R., MAJUMDAR, A., BLACK, L. AND STONE 2, P.J. 1 2

Dept. Biomedical Engineering, Boston University, 44 Cummington St, Boston, 02215, MA, USA Dept. Biochemistry, Boston University School of Medicine, 715 Albany St., Boston, 02118, MA, USA

In diseases, various biochemical processes alter the composition and mechanical properties of soft biological tissues. We developed experimental systems to measure the stress-strain and failure properties of tissues while simultaneously imaging the underlying structure [1]. We measured the stress-strain and failure properties of lung tissues from elastase-treated mice [2] as well as in lung tissues from genetically altered mice that develop spontaneous emphysema [3]. The results provide evidence that failure of collagen contributes to the progressive nature of pulmonary emphysema [4]. We also measured the mechanical and failure properties of elastin rich cell culture based extracellular matrixes [5] with various concentrations of collagen. We found that while increasing the collagen content of the matrix increased its elastic modulus and the failure stress, the failure strain was decreased which is due to the small failure strain of collagen. Some of these matrixes were also digested in the presence of static or cyclic mechanical forces. We found that the presence of mechanical force increased the activity of enzymes and accelerated the rate of decline of the modulus with time during digestion. However, cyclic stretching protected the tissue from deterioration. We developed elastic network models of the lung tissues [3,4,6] and the tissue engineered materials. The network models demonstrate how mechanical force accelerates the rate of decline of the modulus in the presence of digesting enzymes. Using percolation, we also demonstrate how the progression of disease processes such as fibrosis or emphysema can be better understood [6]. Specifically, the global elastic properties of the network can be linked to percolation of local lesions such as clusters of stiff regions in fibrosis or clusters of failed regions in emphysema. Additionally, we will demonstrate how the inverse process of percolation, whereby a percolating cluster is broken down to a set of isolated clusters, can be used as a guiding principle in functionally efficient tissue engineering. References [1] Kononov, S., K. Brewer, H. Sakai, F. S. A. Cavalcante, C. R. Sabayanagam, E. P. Ingenito and B. Suki. Roles of mechanical forces and collagen failure in the development of elastase-induced emphysema. Am. J. Resp. Crit. Care. Med. 164: 1920-1926, 2001. [2] Ito, S., E.P. Ingenito, K.K. Brewer, L.D. Black, H. Parameswaran, K.R. Lutchen, B. Suki. Mechanics, nonlinearity, and failure strength of lung tissue in a mouse model of emphysema: possible role of collagen remodeling. J Appl. Physiol. 98: 503-511, 2005. [3] Ito, S., E. Bartolák-Suki, J. M. Shipley, H. Parameswaran, A. Majumdar, B. Suki. Early emphysema in the tight skin and the pallid mice: roles of microfibril associated glycoprotein, collagen and mechanical forces. Am. J. Respir. Cell Mol. Biol. 34(6):688-694, 2006. [4] Suki, B., K.R. Lutchen, E.P. Ingenito. On the progressive nature of emphysema: Roles of proteases, inflammation, and mechanical forces. Am. J. Resp. Crit. Care. Med. 168: 516-521, 2003. [5] Black, L.D., K. K. Brewer, S. M. Morris, B. M. Schreiber, P. Toselli, M. A. Nugent, B. Suki, and P. J. Stone. Effects of elastase on the mechanical and failure properties of engineered elastin-rich matrices. J. Appl. Physiol. 98: 14341441, 2005. [6] Bates, JHT, GS Davis, A Majumdar, KJ Butnor, and B Suki. Linking parenchymal disease progression to changes in lung mechanical function by percolation. Am. J. Resp. Crit. Care. Med. (In press).

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LECTURE ABSTRACTS Thursday 23rd August

12:00-12:30

Parallel Session b.2.2. Regulatory Systems of Signal Transduction in Airway Smooth Muscle Contraction BRUMEN 1,2, M. AND FAJMUT 1,2, A. University of Maribor, Faculty of Natural Sciences and Mathematics, Medical Faculty, and Faculty of Health Sciences, Slomškov trg 15, SI-2000 Maribor, Slovenia; [email protected] 2 Institute Jožef Stefan, Jamova 39, SI-1000 Ljubljana, Slovenia 1

By the means of mathematical modelling, we shall present calcium signal transduction pathway in contraction of airway smooth muscle cells (ASMC) and discuss the application of the model to asthma diseases. Variation of cytosolic calcium concentration in the form of either single biphasic pulse or oscillation is the primary signal that induces development of force and consecutive contraction in ASMCs. On molecular level, important steps in this calcium signal transduction pathway are phosphorylation and dephosphorylation of myosin light chain (MLC). Phosphorylated MLC enables myosin attachment to actin, triggers myosin cross-bridge cycling and consecutive sliding of myosin filaments relative to actin filaments leading to development of force. A level of contraction and magnitude of force depend on the balance between activities of enzymes MLC- kinase (MLCK) and phosphatase (MLCP) whereby activation/inactivation of MLCK appears to have a key role in decoding of the calcium signal [1]. However, MLCP offers many possibilities of calcium- dependent/independent regulation of force development since it is a target of many different enzymes and signal transduction pathways. Predictions of both enzyme activities and of time dependent force development are analysed with respect to general principles of control action of kinases and phosphatases in signal transduction. Our results confirm the predictions of other kinase/phosphatase systems that kinases primarily regulate the amplitude of a signal whereas phosphatases determine the width and the amplitude of a signal [2]. Simulations of elevated total contents of MLCK and MLCP are discussed in relation to asthma. Namely, cells obtained from asthmatic subjects contain more MLCK in total than normal cells [3]. Cells with greater MLCK content also shorten more and faster than normal ones [3]. Simulations by our model show the same directional changes. Finally, regulation of myosin dephosphorylation is discussed in view of interrelation between calcium signal transduction pathway and inhibition of MLCP by potent inhibitors, whereby arachidonic acid (AA) is one of them. The metabolism of AA in eosinophils and mast cells gives also an insight in aspirin intolerance in asthma (AIA) [4]. References [1] Mbikou, P, Fajmut, A, Brumen, M, Roux, E (2006) Cell Biochem. Biophys. 46: 233-251. [2] Hornberg, JJ, et al. (2005) FEBS Journal 272: 244-258. [3] Ma, X, et al. (2002) Am. J. Physiol. Lung Cell Mol. Physiol. 283: L1181-89. [4] Szczeklik, A and Stevenson, DD (1999) J. Allergy Clin. Immunol. 104: 5-13.

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LECTURE ABTRACTS rd

Thursday 23 August

12:30-13:00

Parallel Session b.2.3. Thermodynamic parameters of actin under different agents and its possible role in the ATP hydrolysis cycle of muscle BELÁGYI J. AND LİRINCZY D. Department of Biophysics, University Pécs, Faculty of Medicine, Szigeti str. 12, Pécs, H-7624, Hungary, [email protected]

Actin filaments the main components of the cytoskeletal structure, are the major constituents of the complex filament network, that is responsible for many vital functions of living cells. The dynamic changes of the actin cytoskeleton are tightly controlled by many regulatory pathways and play important roles in cell motility, endocytosis, phagocytosis and cytokinesis. In all these biological activities the actin filament network needs to be dynamic in a sense, that new actin filaments should be formed and old actin filaments should be decomposed to actin monomers quickly. This dynamic nature of the actin filament network is largely attributed to quick cell responses, which are manifested through the action of actin-binding proteins. The paper will focus on dynamic and conformational adaptation of the actin and actin filaments in the presence of different environmental factors and agents. It is known that nucleotides (ATP, ADP) and nucleotide analogues (ADP.Vi, ADP.AlF4) induce conformational changes in the DSC pattern both in actin filament system and in muscle fibers that contain actin. Nucleotides affect the main protein myosin in fibers, and produce shifts in its transition temperature. It is especially interesting, how it is possible to assign the changes of DSC pattern to the different proteins, and how the dynamic changes of actin can contribute to the muscle activity during ATP hydrolysis. Acknowledgement The SETARAM Micro DSC-II used in the experiments was purchased with funds provided by the National Research Foundation Grant CO-272.

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LECTURE ABSTRACTS Thursday 23rd August

14:00-14:30

Parallel Session a.3.1. EPR spin-trapping of oxygen free radicals in chemical and biological systems: Reevaluation of the role of Fenton reaction GORAN BAČIĆ Faculty for Physical Chemistry, Univ. of Belgrade, Studentski trg 12-16, Belgrade, 11000, Serbia, [email protected]

A brief account of the difficulties involved in the detection of oxygen free radicals in chemical and biological systems is presented accompanied by the rationale for using the EPR spin-trapping technique in such studies. Studies of the ability of EPR spin traps to detect oxygen-centered radicals produced by two generator systems (Fenton reaction and Hypoxantine/Xantine Oxidase reaction) and four biological systems (plant plasma membranes, plant cell wall, islets of Langerhans and human cerebrospinal fluid) are presented. Certain technical aspects of EPR experiments related to the successful trapping of free radicals are also discussed. Comparative analysis of the characteristics of different spin-traps (DEPMPO, EMPO and BMPO) to detect free radicals in terms of adduct stability and the ability of a given spin trap to enable detection of simultaneous production of different free radicals in systems akin to the biological systems is given. The principal outcome of experiments using free radicals generating systems and EPR spin traps is the occurrence of an additional reactive species which was determined to be a hydrogen radical (&H). Although the existence of this radical has been speculated for some time, it has never been proved in Fenton reaction. Since our experiments using various spin traps unambiguously showed the production of &H radical in the Fenton reaction a novel mechanism of the Fenton reaction is proposed. The production of &H was also detected in biological systems under both physiological and pathophysiological conditions. Analyzing various systems using the novel Fenton reaction scheme and EPR spin trapping, it was concluded that under physiological conditions the Fenton mechanism, besides its destructive role in producing harmful &OH radicals, could also have a constructive, regulatory function in the maintenance of the redox equilibrium and/or some other specific function such as conductance of biological signals. These results set the stage for a better understanding of the role of free radicals in a number of conditions such as neurological diseases, sepsis, diabetes and ageing and some of the examples will be discussed in details.

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LECTURE ABTRACTS rd

Thursday 23 August

14:30-15:00

Parallel Session a.3.2. The Physiological Roles of the Native Protochlorophyllide Complexes BÖDDI, B. Department of Plant Anatomy, Eötvös University, Pázmány P. s. 1/c., Budapest, H-1117, Hungary, [email protected]

Protochlorophyllide (Pchlide), the precursor in chlorophyll biosynthesis is organized into various complexes in vivo. The majority of this pigment is built into ternary complexes with protein units of the enzyme NADPH:Protochlorophyllide oxidoreductase (POR) and NADPH. These ternary complexes can be monomers or may aggregate into dimers and oligomers. In etioplasts of leaves of dark-grown seedlings, mainly dimers and oligomers are present, which are flash-photoactive; the photoreduction of Pchlide into chlorophyllide (Chlide) takes place upon ms illumination. These complexes contain surplus NADPH, which have structural role in the aggregate formation but also drive Chlide- and Pchlide- micro-cycles. In these micro-cycles, NADP+ containing Pchlide and Chlide complexes are formed on illumination in which the NADP+ is re-reduced into NADPH cyclically. The photoreceptor function of these complexes has been hypothesised. Etioplasts of stems of many dark-grown seedlings, inner leaf primordia of closed buds, inner leaves of white cabbage and etioplasts in fruit wall of several fruits with dark-green external tissue layers or thick peel, contain Pchlide predominantly in monomer stage. Only a small amount of monomer Pchlide is connected to the monomer units of POR in these tissues. Recently, separate photoreduction of this monomer was observed under 632.8 nm laser light illumination and the artificial aggregation as well as the flash-photoactivity of these aggregates were described in our laboratory. A great amount of this pigment, however, is not built into the above-mentioned ternary complexes. In these tissues, illumination with natural light causes photo-oxidation rather than photoreduction of Pchlide. As a result, all chlorophyllous pigments are strongly bleached; in this case Pchlide monomers behave as photosensibilizer of the photo-oxidative damage. A shortage in NADPH seems to stimulate the bleaching. (Interestingly, the POR content does not change during bleaching; when the bleached samples are regenerated in the dark, the newly synthesised Pchlide accumulate directly in the reaction centres of the POR oligomers.) An oxidation reaction cascade is provoked resulting in the production of ROS. O2-, H2O2 were detected as well as lipid peroxidation in the cells of illuminated tissues. These processes cause wilting and other serious damage in these plant tissues. The photo-damage can be prevented with ascorbate pretreatment of the tissues; consequently, the general redoxi state of the cells is very important. The aggregation of the POR units gives a protection against photo-oxidation. Until the co-factor NADP is in reduced NADPH state, photoreduction or at least redoxi microcycles take place. The POR aggregates are integral components of the prolamellar bodies of etioplasts. The native arrangement of the prolamellar body membranes, POR aggregates and the accumulation of Pchlide and NADPH in leaves, all are very important for effective chlorophyll biosynthesis. The question remains open, why monomer and photosensitive structures have evolved in certain plant tissues. This is a summary of recent results of a project in which the following persons took part: Solymosi, K., Kósa, A., Szenzenstein, A., Erdei, N. and Vitányi, B., (all from the Department of Plant Anatomy of Eötvös University), Kis-Petik, K. and Fidy J., (Semmelweis University, Budapest), Hideg, É. and Barta, Cs. (Plant Biology Institute of the Biological Research Center of H.A.S., Szeged, Hungary), Sundqvist, C. (Göteborg University, Sweden), Franck, F. (University of Liege, Belgium). Acknowledgement We thank the Hungarian Research Foundation for financial support (OTKA TO308003).

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LECTURE ABSTRACTS Thursday 23rd August

15:00-15:30

Parallel Session a.3.3. Thermo-optically induced reorganizations in light-harvesting antennae GARAB, G. Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62, H- 6726 Szeged, Hungary. [email protected]

Earlier we have shown that the main chlorophyll a/b light harvesting complexes of photosystem II (LHCII) form chirally organized macrodomains both in vivo and in vitro. These macrodomains are thought to play important structural and functional roles. Together with stacking, the high lateral self-aggregation (packing) of LHCII and PSII-LHCII supercomplexes explain the lateral segregation (sorting) of the two photosystems between the granum and stroma membranes, and thus the macrodomain organization takes part in the assembly and stabilization of the granum [1]. They also serve the structural basis for long distance migration of the excitation energy. The chiral macrodomains have also been shown to possess a remarkable structural flexibility; most notably, they have been shown to be capable of undergoing light-induced reversible reorganizations. These structural changes are largely independent of the photochemical activity of thylakoids, and are approximately linearly proportional to the light intensity above the saturation of photosynthesis – an important, unique feature with respect to protection of plants against excess excitation [2,3]. Further, isolated, lipid-enriched, loosely stacked lamellar aggregates of LHCII also possess the ability to undergo similar reorganizations. These structural changes are accompanied by fluorescence quenching transients, and, literature data [4,5] show, are also involved in important enzymatic reactions - suggesting their involvement in regulatory processes in excess light. As concerns the nature of the structural changes, we have shown that light induces (i) unstacking of membranes, followed by (ii) a lateral desorganization of the macrodomains, and (iii) monomerization of the LHCII trimers [6]. These structural transitions are accounted for by a novel, biological thermo-optic mechanism: fast thermal transients, arising from dissipated excitation energy, which can lead to elementary structural transitions in the close vicinity of the site of dissipation due to the presence of ‘built-in’ thermal structure-instabilities [7]. Thermo-optically induced structural transitions are thought to lend local structural flexibility to molecular (macro)assemblies of high stability, and appear to operate in the regulation of different antenna systems [8]. Our present investigations are focused on further possible effects of fast, local thermal transients due to dissipation of unused photon excitation in different systems photon dissipation and thermal instabilities. Acknowledgement This work was supported by grants from OTKA (K 63252) and EU FP6 MC RTN (INTRO2). References [1] Mustárdy, L, Garab, G (2003) Trends Plant Sci. 8: 117-122. [2] Barzda, V, Istokovics, A, Simidjiev, I, Garab, G (1996) Biochemistry 35: 8981-8985. [3] Garab, G, Cseh, Z, Kovács, L, Rajagopal, S, Várkonyi, Z, Wentworth, M, Mustárdy, L, Dér, A, Ruban, AV, Papp, E, Holzenburg, A Horton, P (2002) Biochemistry 41: 15121-15129 [4] Zer, H, Vink, M, Keren, N, Dilly-Hartwig, HG, Paulsen, H, Herrmann, RG, Andersson, B, Ohad, I (1999) Proc Natl Acad Sci USA 96: 8277-8282. [5] Yang, DH, Paulsen, H, Andersson, B (2000) FEBS Lett 466: 385-388. [6] Dobrikova AG, Várkonyi Z, Krumova SB, Kovács L, Kostov GK, Todinova SJ, Busheva MC, Taneva SG, Garab G (2003) Biochemistry 42: 11272-11280. [7] Gulbinas, AU, Karpicz, V, Garab, G, Valkunas, L (2006) Biochemistry 45: 9559-9565. [8] Stoitchkova, K, Zsiros, O, Jávorfi, T, Páli, T, Andreeva, A, Gombos, Z, Garab, G (2007) Biochim Biophys Acta – Bioenergetics (in press).

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LECTURE ABTRACTS rd

Thursday 23 August

15:30-16:00

Parallel Session a.3.4. Adaptation of dehydrogenases to extreme environments ISTVÁN HAJDÚ 1, ANDRÁS SZILÁGYI 1, JÓZSEF KARDOS 1,2, PÉTER ZÁVODSZKY 1 1Institute

of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary, of Biochemistry, Eötvös Loránd University, Budapest Hungary, [email protected]

2Department

Dehydrogenases play crucial roles in diverse metabolic processes throughout all biological systems in a wide range of temperatures from the cold of polar regions to the heat of thermal springs. It is assumed that the broad variety of dehydrogenases have a common adaptation strategy for extreme temperatures. The analysis of the temperature dependence of the catalyzed enzymatic reaction offer a possibility to elucidate the strategy. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a hydride transfer enzyme in the glycolytic cycle, and exhibits several diverse roles in living systems. The activity profiles of the catalyzed reactions show non-Arrhenius behaviour, which can be explained by bulk flexibility studies based on H/D exchange experiments. We found that the thermophilic Th. maritima GAPDH is less flexible at room temperature than the mesophilic rabbit GAPDH, while the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a “corresponding state” regarding conformational flexibility. Three orthologous variants of 3-isopropylmalate dehydrogenase (IPMDH) were used to confirm our observations. The van’t Hoff plots for the enzymesubstrate Michaelis constants are highly nonlinear, indicating a temperature dependent change in the mechanism of substrate binding. Local flexibilities were measured by hydrogen/deuterium exchange experiments while relative domain-domain and subunit-subunit motions were characterized by fluorescence resonance energy transfer measurements. Both measurements indicated changes in the nature and range of the fluctuations with increasing temperature. The stability and conformational flexibility of binary and nonfunctioning ternary complexes demonstrate the different mode of substrate and coenzyme binding. This observation supports the idea that conformational fluctuations have a significant role in the catalytic function by regulating enzyme-ligand dynamic interactions causing the unusual activity profile of dehydrogenases.

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LECTURE ABSTRACTS Thursday 23rd August

14:00-14:30

Parallel Session b.3.1. How does the hydrogenase work? BAGYINKA, CS Institute of Biophysics, Biological Research Center, Temesvári krt. 62., Szeged , H-6726, Hungary

Hydrogenases are metalloenzymes that catalyze the reaction H2 ↔ 2H+ + 2e-. Two distinct groups of hydrogenases can be defined depending on the metal content of the protein. There are iron-only and [Ni-Fe] hydrogenases. The [Ni-Fe] enzymes are usually heterodimers involving a small (~30 kDa) and a large (~60 kDa) subunit, giving an overall molecular mass of around 90-100 kDa. The metals are organized into 2-3 FeS clusters and a Ni-Fe binuclear center. The Ni-Fe binuclear center or its ligand environment is believed to be the hydrogen-binding site though the exact binding location has not yet been determined. The FeS clusters transfer the electrons from the Ni-Fe binuclear center to the terminal electron acceptor [1,2]. Although the enzymatic activity of hydrogenase is determined routinely, a number of contradictory results have been published. Despite the many features that have been described in the hydrogenase reaction, the activity of this class of enzymes has not yet been thoroughly explained [3–6]. However, there is a consensus in the hydrogenase literature that the reaction is linear; no feedback from any parts of the reaction occurs, except that some authors admit that there might be a nonlinear feedback during the activation of the enzyme [7–8]. We recently demonstrated that the hydrogenase catalytic reaction includes at least one autocatalytic step [9-11]. This assumption was based on the special patterns of the hydrogenase-uptake reaction in a thin-layer reaction chamber and on the autocatalytic oscillations in the fast absorption kinetics of the methyl viologeninitiated reaction of hydrogenase. The assumption of an autocatalytic step explains most of the contradictory findings in previous publications. In this lecture we will provide further evidences for the autocatalytic nature of the reaction, describe how this behaviour of the enzyme can be affected, and where in the reaction cycle is it possible to include the autocatalytic step. Acknowledgement We are grateful for the financial support of the Hungarian Science Foundation [OTKA T049276 and OTKA T049207] and AUTOESKORT Ltd. References [1] Volbeda, A., M.H. Charon, C. Piras, E.C. Hatchikian, M. Frey and J.C. Fontecilla-Camps. 1995. Nature 373: 580587. [2] Sherman, M. B., E. V. Orlova, E.A. Smirnova, S. Hovmoller and N. A. Zorin 1991. J. Bacteriol. 173: 2576-2580. [3] Fisher, H. F., A. I. Krasna, and D. Rittenberg. 1954. J. Biol. Chem. 209:569–578. [4] De Lacey, A. L., J. Moiroux, and C. Bourdillon. 2000. Eur. J. Biochem. 267:6560–6570. [5] Armstrong, F. A. 2004. Curr. Opin. Chem. Biol. 8:133–140. [6] Kurkin, S., S. J. George, R. N. F. Thorneley, and S. P. J. Albracht. 2004. Biochemistry. 43:6820–6831. [7] Albracht, S. P. J. 2001. Spectroscopy: the functional puzzle.. In Hydrogen as a Fuel: Learning from Nature. R. Cammack, M. Frey, and R. Robson, editors. Taylor and Francis, London, UK; New York, NY. [8] Cammack, R. 2001. Hydrogenases and their activities. In Hydrogen as a Fuel: Learning from Nature. R. Cammack, M. Frey, and R. Robson, editors. Taylor and Francis, London, UK; New York, NY. [9] Bagyinka, C., J. İsz, and S. Száraz. 2003. J. Biol. Chem. 278:20624–20627. [10] İsz, J., and C. Bagyinka. 2005. Biophys. J. 89:1984–1989. [11] İsz, J., Bodó, G., Branca, R.M.M., and C. Bagyinka. 2005. Biophys. J. 89:1957–1964.

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LECTURE ABTRACTS rd

Thursday 23 August

14:30-15:00

Parallel Session b.3.2. Conformation and intermolecular interactions of proteins revealed by high pressure FTIR studies SMELLER, L. Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, [email protected]

Infrared spectroscopy is a useful tool for studying both intra- and intermolecular aspects of the protein structure. Analysing the conformation sensitive amide I band (in the region of 1600-1700cm-1), we can obtain the secondary structure and the specific pair of bands at 1616 and 1685 cm-1 is characteristic for the intermolecular hydrogen bonds, stabilizing protein aggregates. Pressure is a thermodynamic parameter, which is theoretically equally important as the temperature. Still the number of pressure studies is much lower than the one of the investigations as function of the temperature. We used high pressure (up to 1 GPa) to perturb the structure of number of proteins, in order to obtain stability, volumetric and kinetic information. Pressurization has different effects on protein structure, depending on the pressure range used. typically 500 MPa pressure unfolds proteins, destroying the secondary structure. Much lower pressure can already induce disaggregation of protein chains which formed oligomers or certain type of aggregates. We proved that pressure unfolded-refolded proteins can form partially refolded intermediate structures, which have high tendency for aggregation [1]. In the present study we investigated myoglobin, horseradish peroxidase, and hen egg white lysozyme. A systematic investigation of the pressure-temperature phase space led to metastable states on the pressuretemperature plane. Partially folded, aggregation prone metastable states did also form in these pressuretemperature regions. In the next step we investigated the kinetics of the aggregation of these partially folded states. measurement of the kinetical parameters as function of the pressure allowed us to determine the activation volume of the aggregation process. Acknowledgement This work has been supported by OTKA T49213. References [1] Smeller, L. (2002) P Biochimica et Biophysica Acta - Protein Structure et Molecular Enzymology 1595: 11-29.

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LECTURE ABSTRACTS Thursday 23rd August

15:00-15:30

Parallel Session b.3.3. Dynamics and interactions of lipids and proteins in biological membranes. An infrared spectroscopic study. SZALONTAI, B. Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt. 62, 6701 Szeged, P.O.B. 521, Hungary, [email protected]

Maintaining the barrier properties and the functioning (energy production, signal transduction, material transport etc.) of a biological membrane, a given membrane dynamics is required. This dynamics is adapted to the physiological conditions (e.g. temperature, light in photosynthetic organisms, different stresses) of the given membrane. Concerning the barrier properties, the dynamics of membrane lipids and the lipid-protein interactions are thought to be more important. The biological functioning is assured by the dynamics of the membrane proteins, which may depend on the lipid-protein interaction as well. The balance between lipidand protein-dynamics and the role of lipid-protein interaction in maintaining membrane dynamics is still not fully understood. Correlation of lipid disorder with membrane protein dynamics has been studied with infrared spectroscopy, by combining data characterizing lipid phase, protein structure and, via hydrogen-deuterium exchange, protein dynamics. The key element of this approach was a new measuring scheme, by which the combined effects of time and temperature on the H/D exchange could be separated. Two series of infrared difference spectra were created, one representing differences only in time (ISO spectra), the other in time and temperature together (∆T spectra). The changes in these series as a function of temperature were analysed by Singular Value Decomposition (SVD). There is one, general fining of the experiments, which needs further studies and analysis, i.e. the frequency of the amide II band, disappearing upon H/D exchange is always lower by 5-8 cm-1 in the ISO spectra, than in the ∆T spectra. In addition, the lower amide II frequency seems to correlate with the physical state of the membrane lipids. To separate the lipid effects on membrane dynamics, thylakoid membranes of genetically engineered cyanobacteria grown at different temperatures were studied. These thylakoids differed only in the fatty acid composition of their lipids. To reveal the generality of the found correlations between lipid and protein dynamics, rat liver mitochondrium and yeast vacuolum membranes were also studied. In dissolved lysozyme, the H/D exchange, as a function of temperature, involved only reversible movements (the secondary structure did not change considerably); heat denaturing was a separate event at distantly higher temperatures. In contrast, in membrane proteins, enhanced H/D exchange rates always led to denaturing. In the cyanobacterial thylakoid membranes, at the high-temperature end, membrane proteins exhibited two separated temperature ranges where both the H/D exchange rate and the protein secondary structure altered considerably. The lower temperature range of changes (58-66 oC) involved only proteins and was not sensitive for the growth conditions. In the higher temperature range of changes both proteins and lipids were involved. The actual temperatures of these ranges reflected the difference of the growth temperatures; they were 75-85 oC for the cells grown at 25 oC, and 80-90 oC for the cells grown at 35 oC. In mitochondrial membranes, which have much more unsaturated lipids, and consequently a higher lipid disorder at the same temperatures as compared to that of cianobacterial thylakoids, proteins exhibited one single transition from the native to the denatured state, but the nature of the changes was the same.

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LECTURE ABTRACTS rd

Thursday 23 August

15:30-16:00

Parallel Session b.3.4. Amide Hydrogen/Deuterium Exchange in Sphingomyelin/Cholesterol Membranes Studied by ATR-FTIR Spectroscopy ARSOV 1, Z., QUARONI 2, L. 1 2

Laboratory of Biophysics, "Jožef Stefan" Institute, Jamova 39, 1000 Ljubljana, Slovenia, [email protected] Mid-IR Beamline, Canadian Light Source, 101 Perimeter Rd, Saskatoon (SK), Canada, S7N 0X4

The amide hydrogen/deuterium (H/D) exchange studied by Fourier transform infrared (FTIR) spectroscopy and assessed with time of exposure to deuterated-water-based solvent contains valuable information about the protein dynamics and structure [1]. The H/D exchange studies of amide group in lipids are not so common, although valuable information about intermolecular interactions can be gained [2]. A major current issue in membrane biophysics is the incidence and stabilization of lateral structure of biological membranes. Interesting model systems are lipid mixtures exhibiting the so called liquid-ordered phase, which in some respect resembles the cholesterol-rich lipid domains in biological membranes. The major components of these domains are thought to be sphingomyelin (SM) and cholesterol (Chol). It was implied that SM-Chol interactions are strengthened by hydrogen bonding [3]. For example, molecular dynamics simulations show that a hydrogen bond between cholesterol oxygen and hydrogen of the NH group of SM is formed (among other possible hydrogen bonds) [4]. Our aim was to check whether hydrogen bonding in SM/Chol system affects the kinetics of the amide H/D exchange. Attenuated total reflection (ATR) FTIR spectroscopy was used because it is well suited for experiments with samples prepared in excess (deuterated) water due to small penetration depth of the evanescent field. In addition, owing to the multibilayer lipid structure formed on the internal reflection element the absorption due to water is minimized, while the absorption due to lipids is maximized. And finally, the addition or removal of different solvents during the experiment is simple. The results show that the amide H/D exchange is faster in samples containing cholesterol, contrary to the expected restriction of the H/D exchange due to the presence of the SM-Chol hydrogen bond. On the other hand, the effect of the introduction of Chol in the SM membrane is also a decrease in the number of SM-SM intermolecular hydrogen bonds, which also involve hydrogen of the NH group of SM [4]. Consequently, our finding can be interpreted as an indication that the influence of the weakening of the SM-SM intermolecular interaction exceeds the effect of the formation of SM-Chol hydrogen bonds. Acknowledgement This work was carried out with the financial support of the Sincrotrone Trieste and the Slovenian Research Agency. References [1] Tatulian, SA (2003) Biochemistry 42: 11898-11907. [2] Müller, E, Blume, A (1993) Biochim. Biophys. Acta 1146: 45-51. [3] Ohvo-Rekilä, H, Ramstedt, B, Leppimäki, P, Slotte, JP (2002) Prog. Lipid Res. 41: 66-97. [4] Khelashvili, GA, Scott, HL (2004) J. Chem. Phys. 120: 9841-9847.

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LECTURE ABSTRACTS FRIDAY, 24TH AUGUST Friday 24th August 8:30-9:05 Plenary Lecture 4.1. Single-molecule stochastic sensing using nanopores MOVILEANU, L. Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, USA, [email protected]

We have developed a new methodology to examine single polymer dynamics within a protein nanopore, a simple system that is highly relevant to several more complex biological processes such as the translocation of nucleic acids and polypeptides through transmembrane pores. We have used rational design and chemical modification of the α-hemolysin (αHL) protein to devise unusual nanostructures with movable polymeric arms. The ionic current through a single protein nanopore was determined by single-channel electrical measurements in lipid bilayers. The results revealed unprecedented details of polymer behavior at the singlemolecule level. In addition, a variety of stochastic sensing devices for small molecules or macromolecular analytes were derived. For example, we tethered a single oligonucleotide within the same nanopore to allow DNA duplex formation inside the pore lumen. Recently, temperature-responsive protein pores have been designed by engineering a single polypeptide within the large vestibule of the channel. Moreover, binding sites engineered within strategic positions of the αHL pore permitted the conversion of this protein from a blank state for protein translocation into a β-barrel protein translocase with predefined biophysical properties, such as ionic selectivity and polypeptide permeation. Acknowledgements This work is funded by the Keck Foundation, the National Science Foundation, and the Syracuse University start-up funds.

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LECTURE ABTRACTS th

Friday 24 August

9:05-9:40

Plenary Lecture 4.2. Electron transport through single redox metalloproteins: what scanning probe techniques can teach us FACCI, P. CNR-INFM-S3, Via G. Campi, 213/A., Modena, I-41100, Italy, [email protected]

The transfer of electrons in biological systems represents the mean by which a relevant number of complex functions is partially or fully accomplished. Key phenomena such as respiration, photosynthesis, catalytic reactions, etc. involve, as a crucial step, the transfer of one or more electrons between molecular partners along free energy cascades. A peculiar role in biological electron transfer is played by special classes of biomolecules called redox metalloproteins. Redox or electron transfer metalloproteins are characterized by bearing one or more metal ions in their active sites, whose oxidation state can reversibly change, being thus at the basis of their ability to exchange electrons with molecular partners. In this contribution I will show the results that have been achieved in a paradigmatic case (the blue copper protein Azurin from Pseudomonas aeruginosa) by the use of a particular scanning probe technique (the electrochemical scanning tunnelling microscope - ECSTM) whose operation is customized to the peculiar conditions required for studying electronic transport via a single metalloprotein. The redox state of Azurin, chemisorbed on a gold surface, which plays the role of working electrode of an electrochemical cell, can be externally controlled, and STM imaging as a function of the molecular redox state can be performed. By this approach, which requires a special STM set-up involving tip insulation and the use of a bipotentiostat, important information on the molecular electronic functioning can be achieved. Namely, the role of the particular metal ion at the active site has been elucidated [1,2], along with the particular physical mechanism ruling redox state-mediated electron transfer in this metalloprotein [3]. Finally, future trends in scanning probe microscopy instrumental development towards empowering this kinds of studies will be accounted for [4,5]. References [1] A. Alessandrini, M. Gerunda, G. Canters, M. Ph. Verbeet, P. Facci (2003) Chem. Phys. Lett. 376: 625. [2] A. Alessandrini, M. Salerno, S. Frabboni, P. Facci (2005) Appl. Phys. Lett. 86:133902. [3] A. Alessandrini, S. Corni, P. Facci (2006) Phys. Chem. Chem. Phys. 8:4383. [4] A. Alessandrini, P. Facci (2005) Meas. Sci. Technol. 16: R65. [5] C. Menozzi, A. Alessandrini, G. Gazzadi, P. Facci (2005) Ultramicroscopy 104: 220.

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LECTURE ABSTRACTS Friday 24th August

9:40-10:15

Plenary Lecture 4.3. Optical Micromanipulations of the Intracellular Design TOLIC-NORRELYKKE, I.M. Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, 01307, Germany

How does a cell organize its interior? Spatial organization is crucial for life at all levels of complexity, from macromolecules to cells, organs, and organisms. At the level of single cells, segregation of genetic material and a correct cell division relies on proper cell shape and position of organelles inside the cell. Microtubules and the cell nucleus play a key role in organizing the intracellular space. A direct way to study the organization and positioning of organelles employs mechanical perturbation of intracellular structures. For this purpose, we developed a combined system for nonlinear microscopy, laser nanosurgery, and optical trapping. The system enabled us to visualize and dissect single microtubules and mitotic spindles, as well as to displace the cell nucleus. By applying these techniques to fission yeast cells, we show that the force for mitotic spindle elongation is generated in the spindle midzone, while astral microtubules help to align the spindle by pushing against the cell edge. Furthermore, during interphase, microtubule pushing against the cell tips centers the nucleus. The centrally placed nucleus, in turn, positions the cell division plane precisely at the cell equator. We propose a model where microtubule pushing forces are responsible for different aspects of cell symmetry throughout the fission yeast cell cycle.

Fig. 1. Displacing the cell nucleus by optical tweezers.

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LECTURE ABTRACTS th

Friday 24 August

10:15-10:50

Plenary Lecture 4.4. A Molecular Model of Rheological Behavior of Living Cells ROSENBLATT 1, N., ALENCAR 2, A. M., MAJUMDAR 3, A., SUKI 1, B., AND STAMENOVIĆ 1, D. Department of Biomedical Engineering, Boston University, 44 Cummington St., Boston, MA 02215, USA, [email protected] Harvard School of Public Health, 665 Huntington Av., Boston, MA 02115, USA 3 Department of Physics, Boston University, Street No., Town, Postal Code, State 1 2

Material behavior of adherent cells is characterized by two distinct features: a) material moduli scale with time/frequency with a power-law [1]; and b) cell stiffness increases with increasing cytoskeletal prestress [2]. These phenomena have been explained by mechanisms that are unrelated; the power-law behavior has been explained by soft glass rheology [1], whereas the dependence on the prestress has been explained by stresssupported structures [3]. However, experiments have shown that the power-law rheology and the prestress are associated in living cells and that the power-law exponent (α) decreases with increasing prestress [2]. Since α is an index of transition between elastic and viscous behaviors, these findings suggest that the prestress regulates cell’s viscoelastic behavior. We explain these observations as follows. The rheological behavior of the cytoskeleton (CSK) must reflect the dynamics of its actin molecules. These are semiflexible polymer chains that fluctuate under thermal agitations. Under prestress, they become taut and thus their thermally-driven conformational changes become hampered. Consequently, the CSK stiffens increases and α decreases. Based on this description, we propose a stochastic rheological model of a single semiflexible polymer chain under sustained tension [4]. The chain is composed of nonlinearly elastic, hardening bonds connected by linearly elastic joints. In order to simulate the creep response, the chain is stretched by a constant force (F). To mimic the effect of prestress, we increase F stepwise, in equal steps (δF), such that the prestressing force F0 = F – δF. Monte Carlo simulations show

an initial fast creep, followed by a long power-law creep, and a steady state (Fig. 1A) such that α decreases with

increasing F0 (Fig. 1B), consistent with observations in cells. This behavior is related to the propagation of free energy through the chain in response to stretching, where the propagation speed is regulated by prestress via chain’s nonlinear elasticity. This, in turn, suggests that the entropic dynamics of the chain alone cannot account for the influence of the prestress on cell rheology, and that the internal energy contributions, which result from the nonlinear elasticity, are essential. Our findings imply that the observed complexities of cell rheology exist at the level of individual polymer chains of the CSK. Thus, our approach constitutes a departure from the general thought that cytoskeletal network properties play a primary role in cell rheology.

Fig. 1. A) Chain’s creep curves for different levels of prestress F0; 〈δL〉 = mean change in chain’s length, t = time. B) Power-law exponent α vs. F0.

References [1] Fabry B, Maksym GN, Butler JP, Glogauer M, Navajas D, Fredberg JJ (2001) Phys Rev Lett 87: 148102. [2] Stamenović D, Suki B, Fabry B, Wang N, Fredberg JJ (2004) J Appl Physiol 96: 1600-1605. [3] Stamenović D, Coughlin MF (1999) J Theor Biol 201: 63-74. [4] Rosenblatt N, Alencar AM, Majumdar A, Suki B, Stamenović D (2006) Phys Rev Lett 97: 168101.

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LECTURE ABSTRACTS Friday, 24th August 11:30-12:00 Parallel Session a.4.1. Nanotechnology with amyloid fibrils MIKLÓS S.Z. KELLERMAYER 1, ÁRPÁD KARSAI 1, LÁSZLÓ GRAMA 1, ÜNIGE MURVA I1, MARGIT BENKE 1, KATALIN SOÓS 2, AND BOTOND PENKE 3 1Dept.

Biophysics, University of Pécs, Faculty of Medicine, Szigeti út 12. Pécs H-7624 Hungary; [email protected] and Nanostructured Materials Research Group of the HAS, Szeged, Dóm tér 8. H-6720 Hungary 3Dept. Medicinal Chemistry and Protein Research Group of the HAS, University of Szeged, Szeged, H-6720 Hungary 2Supramolecular

Amyloid fibrils are pathogenic agents in neurodegenerative and protein misfolding diseases. Because modified amyloid peptide subunits can be generated by synthetic methods and the assembled nanometerscale fibrils are stable under diverse conditions, amyloid fibrils have been suggested for nanotechnology applications [1-4]. However, the necessary well-controlled oriented growth of amyloid fibrils has not been accomplished. In the present work we analyzed the properties of amyloid beta 25-35 (Aß25-35) fibrils for their potential use in nanotechnology. We found that Aß25-35 forms trigonally oriented fibrils on mica. Oriented binding depended on an apparently cooperative interaction of a positively-charged moiety on the Aß25-35 peptide with the K+-binding pocket of the mica lattice. Time-lapse in situ AFM revealed that the formation of oriented fibrils is the result of epitaxial polymerization rather than binding of already assembled fibrils from solution. By varying K+ concentration the growth rate and the mesh size of the oriented amyloid fibril network could be tuned. To utilize Aß25-35 fibrils as chemically active tracks, we prepared fibrils from a mutant peptide in which Asn27 was replaced with Cys (Aß25-35_N27C). We observed that the Aß2535_N27C peptide formed oriented tracks resembling those formed from the wild type peptide. The oriented growth of Aß25-35 fibrils allowed us to examine the detailed dynamics of amyloid assembly. We implemented a simply modified application of the AFM, called scanning force kymography, to monitor the assembly, on mica, of individual A25-35 fibrils with near-subunit spatial and subsecond temporal resolution. Fibril assembly was polarized and discontinuous. Bursts of rapid, concentration-dependent growth phases that extended the fibril by ~8 nm or its integer multiples were interrupted with pauses. The growing fibril may thus fluctuate between a fast-growing and a kinetically trapped blocked state. In sum, the controlled, oriented assembly of Aß25-35 fibrils could be utilized in nanotechnology applications such as forming oriented tracks for molecular devices and generating nanoelectronic circuits. Acknowledgements This work was supported by grants from the Hungarian Science Foundation (OTKA T049591 and TS 049817), and the Hungarian National Office for Research and Technology (OMFB-01600/2006, OMFB-01627/2006, OMFB00198/2007, KFKT-1-2006-0021, RET 08/2004) References [1] Gazit, E. (2006) FEBS Journal 274: 317-322. [2] Hamada, D., I. Yanagihara, and K. Tsumote (2004) Trends in Biotechnol 22: 93-97. [3] Reches, M. and E. Gazit (2003) Science 300: 635-627. [4] Scheibel, T., R. Parthasarathy, G. Sawicki, X.M. Lin, H. Jaeger, and S.L. Lindquist (2003) Proc Natl Acad Sci U S A 100(8): 4527-32.

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Friday, 24 August

12:00-12:30

Parallel Session a.4.2. 3D movie of living fibroblasts MAURER, C., JESACHER, A., SCHWAIGHOFER, A., FÜRHAPTER, S., BERNET, S., RITSCH-MARTE, M. Section for Biomedical Physics, Innsbruck Medical University, Müllerstraße 44, Innsbruck, 6020, Austria

A challenging task in microscopy is the imaging of phase samples. Several solutions have been found in the past. We present a new method for efficient edge enhancement of phase samples. In spiral phase contrast microscopy the Fourier transform of an object is multiplied with a spiral phase function ei*phi, where phi is the spatial angle of the Fourier components. The process is equal two a 2D – Hilbert transformation, enhancing the edges of a phase sample. The singularity in the centre of the spiral phase plate is replaced by a constant disk with diameter on the order of some wavelengths. Therefore the zero order passes through the phase plate without filtering and can be used as reference wave. The interference pattern depends on the phase difference between the zero order and the phase of the spiral phase plate. Shifting the relative phase between the zero order and the phase plate three times by 120 degrees generates three independent interference patterns, which can be added to calculate the amplitude and phase of the sample (Fig.1). The horizontal imaging path of a standard microscope is used to filter the light field. A spatial light modulator (SLM) is put into the Fourier plane of the object. The SLM is steered online by a Computer, which allows fast rotation of the spiral phase hologram. This setup allows us to take 3D images with a frame rate of 20 Hz. We Fig. 1. Phase profile of erythrocytes. Scale in x and y direction in µm, thickness of the cells in arbitrary demonstrate the method with living erythrocytes units. and fibroblasts.

Acknowledgement This work was supported by the Austrian Science Foundation (FWF) Projects No. P18051-N02 and P19582-N20.

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LECTURE ABSTRACTS Friday, 24th August 12:30-13:00 Parallel Session a.4.3. Observation of confined nondiffusive Brownian motion of an isolated particle JENEY, S., LUKIČ, B. AND FORRÓ, L. Institut de Physique de la Matière Complexe, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, [email protected]

Diffusion governed by Brownian motion is an efficient transport mechanism on short time and length scales. Even a highly organized system like a living cell relies in many cases on the random Brownian motion of its constituents to fulfill complex functions. A Brownian particle will rapidly explore a heterogeneous environment that in turn strongly alters its trajectory. Thus, detailed information about the environment can be gained by analyzing the particle’s trajectory. The thermal position fluctuations of a single micron-sized sphere immersed in a fluid were recorded by optical trapping interferometry with nanometer spatial and microsecond temporal resolution. The optical trap has a twofold function: it ensures that the particle remains within the detector range, and it provides a light source for the position detection. We find, in agreement with the theory of Brownian motion including hydrodynamic memory effects, that the transition from the ballistic to the diffusive motion is delayed to significantly longer times than predicted by the standard Langevin equation. This delay is a consequence of the inertia of the fluid and on the shortest time scale investigated, we can even detect the effect of the sphere’s inertia [1]. Furthermore, our study gives insight on the particle’s behavior, when confined by a harmonic potential and also by the presence of a wall. Surprisingly the hydrodynamic memory effects coming from the inertia of the fluid and the harmonic potential act at the same time scale for the studied system [2]. References [1] Lukic, B, Jeney, S, Tischer, C, Kulik, A J, Forro L, Florin, E-L (2005) PRL 95: 160601. [2] Lukic, B, Sviben, Z, Florin, E-L, Kulik, A J, Jeney, S, Forro (2007) submitted to PRE

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Friday, 24 August 11:30-12:00 Parallel Session b.4.1. Synthetic biology and light activated bio-molecular machines RAYMOND SPARROW CSIR – Biosciences, Pretoria, Republic of South Africa, [email protected]

The talk focuses on a new thematic research area, the development of controllable light-activated Biomolecular machines and devices. The presentation gives an overview of the main components of the biologically based machine designed using the principles of synthetic biology. Synthetic biology uses an engineers approach to biological materials and processes at the molecular level. The biological principles of photosynthesis, in-situ production of ATP and motile protein mechanical movement on which the device is based are described. The architecture, functional concepts and some potential applications are outlined. The talk will emphasise the need for an integrated approach to conducting this type of research.

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LECTURE ABSTRACTS Friday, 24th August 12:00-12:30 Parallel Session b.4.2. Optical transitions in M-DNA OMERZU, A. Jozef Stefan Institute, Jamova 39, Ljubljana, 1000, Slovenia

We present measurements of optical absorption and emission spectra of M-DNA, a new form of DNA where divalent metal cations are incorporated into the DNA structure by replacing one of the hydrogens from hydrogen bonds in the interior of double helix. The aim of our work was to investigate how intercalation of Zn2+ ions into the DNA double helix alters the DNA electronic structure and how it reflects in its optical absorption and emission spectra. In the first approximation, the optical absorption spectrum of DNA could be considered as a sum of the absorption spectra of the constituent nucleobases: adenine, guanine, thymine and cytosine. The absorption maxima corresponding to their first π - π* transitions are centred around 4.6 eV. They are blue shifted from the 0-0 transition line due to the Frank-Condon effect. However, a closer look on the absorption spectrum could reveal significant differences which reflect interactions between nucleobases stacked in a double helix structure of DNA. Generally, interactions between chromophores, either of the same kind or different, could be divided into Coulombic and short range interactions. The leading term in the Coulombic interactions is the dipole-dipole one and it is usually the only term considered in the excitonic model of interchromophore electronic interactions and excitation transfer. The dipole-dipole interactions depend on mutual orientations of interacting dipoles. In the case of DNA, strong π - π* transition dipole moments are polarized in a plane of the nucleobases i.e. perpendicular to the DNA molecular axis. The short range interactions - the charge density interpenetration and the quantum mechanical exchange interaction, both depend on interchromophore orbital overlap. They play only a minor role in the DNA structures due to a weak orbital overlap but, when present, they would cause a red shift of the absorption spectrum In our experiment we have used Zn2+ cations which readily form a complex with DNA in alkaline buffers (pH 9). The level of DNA saturation with Zn2+ was controlled by an ethidium bromide (EB) fluorescence assay: Namely, when intercalated into DNA, EB fluoresces about 25 times more strongly than free in solution. Insertion of Zn2+ into the DNA structure precludes the EB intercalation thus making the EB fluorescence a direct measure of percentage of (un)occupied intercalation sites. By measuring optical absorption spectra of Zn-DNA in a solution and in a dry form and comparing them with corresponding spectra of pristine DNA we have found that the absorption maximum shifts to lower energies by ~0.1 eV. Absorption spectra of wet and dry Zn-DNA are almost identical and show a red shift of absorption maxima. The red energy shift indicates an increase of the π - π overlap of adjacent molecular orbitals in a more compact M-DNA structure. We argue that the shift is caused by structural changes in M-DNA double helix induced by the intercalated metal cation. Those structural changes enhance the orbital π - π overlap between molecular orbitals of neighbouring nucleobases and consequently increase the exchange interaction between them. References [1] Lee, JS, Latimer, LJP, Reid, RS (1993) Biochem. Cell. Biol. 71: 162-168. [2] Omerzu, A, Mihailovic, D, Anzelak, B, Turel, I (2007) to appear in Phys. Rev. B.

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Friday, 24 August 12:30-13:00 Parallel Session b.4.3. An atomistic model of DNA: low frequency vibrations and base-pair opening FRANCI MERZEL 1, MARK R. JOHNSON 2 1 2

National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia, [email protected] Institut Laue Langevin, BP156, 38042 Grenoble Cedex 9, France

Understanding the function of biological molecules has evolved from being structure-based to including knowledge of molecular dynamics. In the case of DNA, a key point of interest is base-pair opening as such dynamics are expected to play a key role in replication, transcription and denaturation. Since biological activity takes place at or close to room temperature, the focus tends to be on low frequency vibrations of biological molecules. Atomistic force fields and molecular dynamics simulations for biological molecules are widely used to understand the behavior of such complex systems. For DNA, the CHARMM force field and MD code has been shown to reproduce average, structural properties of different forms of DNA, but it has not been applied to study phonons and molecular vibrations. We have done precisely this to obtain an atomistic picture of the vibrational modes of DNA. In order to compare our atomistic B-DNA model with the INS and IXS phonon data, we have developed a new software package that extends the functionality of a code like PHONON to arbitrarily large systems and extends the gamma point only analysis already available for larger systems to include k-vector dependence. The software reads a Hessian matrix of force constants, constructs and diagonalises the dynamical matrix for any k-vector and calculates spectral observables for coherent and incoherent scattering. The calculated dispersion curve at low frequency for B-DNA is compared with published results and over the whole frequency range (up to 4000 cm-1), the calculation is compared with new, incoherent, inelastic neutron scattering spectra. These comparisons show the model to have good accuracy. The model is therefore exploited to investigate the long sought after, atomistic description of the vibrational modes of DNA. Various simplification schemes are presented for analysing vibrational modes, which are otherwise too complicated to understand in very large systems. References [1] Merzel F, Fontaine-Vive F, Johnson MR (2007) Comp Phys Comm, in press

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LECTURE ABSTRACTS SATURDAY, 25TH AUGUST Saturday, 25th August

8:30-9:05

Plenary Lecture 5.1. Protein structure is determined by the sequence via subset amino acid compositions ISTVÁN SIMON Institute of Enzymology, Karolina 29, Budapest, H-1113 Hungary, [email protected]

While protein sequences are encoded in nucleic acid sequences in a well-known simple form, the structural properties of proteins are coded in their amino acid sequence in a yet unknown way. In the past decades many unsuccessful attempts tried to decipher the later relationship. In this review we argue that the 1D structure of the polypeptide does not directly determine the 3D properties of the protein, rather the connection occurs in a more complex, hierarchical manner via multiple steps. These intermediate stages are governed by composition of distinguished set of residues, segments or sets of segments. In my lecture I will discuss evidences of the hypothesis that structural properties of the polypeptide chain are determined via the composition of various subsets. Thus we propose that the composition serves to bridge the gap between 1D and 3D structure information of proteins.

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Saturday, 25 August

9:05-9:40

Plenary Lecture 5.2. Modeling global properties of proteins LUČIĆ, B. Ruñer Bošković Institute, Bijenička 54, Zagreb, Hr-10000, Croatia, [email protected]

Modeling global structural properties of proteins (like folding types, secondary structure content, folding or unfolding rates, location of a protein in the cell, etc.) from the structure is one of the most important challenges of computational structural biology and biophysics, and the first step is analysis of properties of a new protein sequence. There have been many attempts to predict global protein features, but a lot of models that include many non-significant parameters have been developed and published. Such models are not of high accuracy, especially not of such high accuracy as it was presented in original publications. I will illustrate the overfitting problems in modeling global features of proteins on examples related to modeling protein folding rates [1,2] and protein secondary structure contents [3], as it was pointed out in ref. 4. Folding and unfolding rate constants are modeled by using average of physical/chemical properties of amino acid residue of protein [1,2]. The authors selected many parameters in models comparing with the total number of proteins in data sets. Due to this reason, the correlation in developed models are really due to the chance, and although statistical parameters of fit and leave one out procedure (each compound was removed from the set, model is developed on n-1 compound, and by such a model rate constant for removed protein was 'predicted') 'are' excellent, they are the consequence of the random correlation. To illustrate this, we re-calculated model parameters for 10 proteins of mixed class (eq. 4 in ref. 1) using four parameters (polarity, refractive index, solvent-accessible surface area upon unfolding, and unfolding entropy change of hydration) each having three decimal places for each protein, and obtained exactly the same model parameters (ln(ku) = -67.07·P – 253.53·Rf + 701.48·∆ASA – 506.66·(-T∆Sh) + 49.41; correlation coefficient r = 0.994). But, after that we used the same parameters in which each value was rounded to two decimal places, and model parameters were drastically changed (ln(ku) = -67.07·P – 253.53·Rf + 701.48·∆ASA – 506.66·(-T∆Sh) + 49.41), as well as statistical parameters (r = 0.885). In the final model for unfolding rate constants for all classes (29 proteins) 16 parameters were selected (eq. 5 in ref. 1), what is unambiguous indication that the model is overfitted. The same case is for all other models developed in refs 1 and 2. Improvement of models for folding rate constants by inclusion of novel parameters that are based on properties of amino acid residues and their distribution through sequence will be presented. Second example is related to modeling the protein secondary structure content on four data sets having 166, 262, 398 and 475 soluble proteins [3]. Developed model in ref. 3 involved 57 independent parameters (optimized constants) for all three secondary structure types (α, β and coil) in linear and 247 optimized parameters in nonlinear models. By performing selection of small number (only five) of most important parameters (among 20 frequencies of amino acid residues and 210 frequencies of products of frequencies, and among them product of ala x leu was most important ones), I selected much simpler and better models. Mean absolute error for data set of 262 proteins for three secondary structure contents is 9% with the model having only five parameters for each of three secondary structure types, comparing with corresponding error of 11% obtained in ref. 3. These models can be improved by inclusion of autocorrelation functions that are computed using relevant properties of amino acid residues for each protein sequence, what will be illustrated. Acknowledgement We are grateful to Frank Eisenhaber for providing four data sets. This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (grant 098-1770495-2919). References [1] Gromiha, MM, Selvaraj S, Thangakani AM (2006) J Chem Inf Model 46: 1503-1508. [2] Gromiha MM, Thangakani AM, Selvaraj S (2006) Nucleic Acids Res 34: W70-W74. [3] Eisenhaber F, Imperiale F, Argos P, Frömmel C (1996) Proteins 25:157-168. [4] Lučić B, Piližota T, Trinajstić N (2004) J Chem Inf Comput Sci 44: 113-121.

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LECTURE ABSTRACTS Saturday, 25th August

9:40-10:15

Plenary Lecture 5.3. Studying the extremes of biomolecular dynamics in silico: from protein folding to structure determination BOJAN ZAGROVIC 1, JAN LIFPERT 2, ERIC J. SORIN 2, IAN S. MILLETT 2, SEBASTIAN DONIACH 2, WILFRED F. VAN GUNSTEREN 1, VIJAY S. PANDE 2 1 2

ETH Zurich, HCI G227, Zurich, 8093, Switzerland Stanford University, Stanford, CA, 94305, USA

Biomolecules move, fluctuate and change structure. Indeed, it is precisely their dynamics that bridges structure with function. While experimental approaches are making breathtaking steps forward, currently the only way to probe biomolecular dynamics on a single molecule level and with all-atom, femtosecond resolution is through computer simulation. In this presentation, I will describe some of our recent advances in the area of molecular dynamics simulations with focus on the three principal challenges driving the field forward: 1) how do we accurately represent biomolecules and their dynamics in silico; b) how do we reach the experimentally relevant time scales in our simulations; and 3) how do we adequately link our theoretical predictions with experimental results. In particular, I will focus on using distributed computing "grid" approaches to study protein folding (Folding@Home project), and demonstrate how they have been used to successfully simulate the folding process of several single-domain proteins in atomistic detail [1]. Second, I will discuss how a synergy between simulation and small-angle X-ray scattering (SAXS) and circular dychroism experiments has been used to study the properties of the unfolded state of proteins [2]. Specifically, I will focus on the properties of the polyproline type II (PPII) helix, which has recently emerged as the dominant paradigm for describing the Fig. 1. Distribution of Folding@Home volunteers around the world. For more information visit: conformation of unfolded proteins. Using the distributed folding.stanford.edu computing techniques and seven different force fields we have simulated a short polyalanine chain, which according to NMR experiments should adopt a PPII helix, and compared and contrasted our results with experiment. Finally, I will demonstrate how computer simulations, with their access to microscopic details inaccessible to experiment, can sometimes deliver a qualitatively new interpretation of experimental data on the example of fiber diffraction and helical diffraction theory [3]. I will give examples of fiber diffraction patterns calculated for two different randomwalk models of polymers, with no built-in helical features, giving layer-line intensities characteristic of fiber diffraction patterns from helices with an integral number of units per turn. Acknowledgements. This work has been supported by HHMI and EMBO fellowships, as well as research grants from NIH (USA) and NCCR (Switzerland). References [1] Pande, VS et al. (2002) Biopolymers 68: 91-109. [2] Zagrovic B et al. (2005) PNAS 102: 11698-11703. [3] Zagrovic B (2007) Molecular Physics (submitted)

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LECTURE ABTRACTS th

Saturday, 25 August

10:15-10:50

Plenary Lecture 5.4. Protein-Protein Binding Sites Prediction using Molecular Modeling Approach JANEZ KONC, NEJC CARL, AND DUŠANKA JANEŽIČ National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia, [email protected]

Molecular modeling is indispensable in theoretical research in chemistry, molecular physics, structural biology, new materials development, and other fields. To productively use molecular modeling methods, their theoretical basis must be understood and the appropriate method must be chosen for solving a given problem. A new algorithm to predict protein-protein binding sites using conservation of both protein surface structure and physical-chemical properties in structurally similar proteins is developed. Binding-site residues in proteins are known to be more conserved than the rest of the surface, and finding local surface similarities by comparing a protein to its structural neighbors can potentially reveal the location of binding sites on this protein. This approach, which has previously been used to predict binding sites for small ligands, is now extended to predict protein-protein binding sites [1-4]. The algorithm was tested in predicting protein-protein binding sites on a set of protein complexes from the literature. Each protein complex in the set is split into its constituent chains, and one chain from each complex is compared with one or more of its structural neighbors. The surface that we find to be conserved in both the chain and its neighbor structure(s) is then predicted to be the binding site for the second chain in the complex. To verify the predicted binding site, we compare it against the actual binding site. Examples of binding-site predictions for a set of proteins, which have previously been studied for sequence conservation in protein-protein interfaces, are given. The predicted binding sites and the actual binding sites are in good agreement. Our algorithm for finding conserved surface structures in a set of similar proteins is a useful tool for the prediction of protein-protein binding sites. Our algorithm differs from others in that only the structure of a protein and a couple of its structural neighbors is needed. This approach may give more unbiased predictions of protein-protein binding sites than predictions obtained by other methods, which are trained on a set of existing interfaces. Since it uses a different paradigm, our approach may be best when used in combination with these methods. Our algorithm can also be used to reduce the search space of docking algorithms and can provide new targets for potential inhibitors of protein-protein interactions. Acknowledgement The financial support through grants P1-0002 of the Ministry of Higher Education, Science, and Technology of Slovenia is acknowledged. References [1] Konc, J.; Hodošček, M.; Janežič, D.; (2006) Croat.Chem. Acta 79: 237-241. [2] Konc, J.; Janežič, D. (2007) A Maximum Clique Problem Revisited. (submitted). [3] Konc, J.; Janežič, D. (2007) Lect. Notes Comput. Sci. 4432: 399-406. [4] Konc, J.; Janežič, D. (2007) J. Chem. Inf. Model. 47: 940-944.

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LECTURE ABSTRACTS Saturday, 25th August

11:30-12:00

Plenary Lecture 6.1. Global conformational changes, cavities, ligand binding and migration in proteins PERAHIA 1, D., MARECHAL 2, J-D., MOUAWAD 3, L., FLOQUET, N.4 AND ROBERT 1, C. Modélisation et Ingénierie des Protéines, IBBMC, Bât 430, Université Paris-Sud, 91405 Orsay, France, [email protected] Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain 3 Inserm U759, Institut Curie-Recherche, Bâtiment 112, Université Paris-Sud, 91405 Orsay cedex, France 4 Laboratoire de Biochimie Médicale et Biologie Moléculaire, CNRS UMR 6198, Université de Reims-Champagne-Ardenne, France 1 2

A cornerstone of molecular simulations is the exploration of large amplitude conformational changes of macromolecules in order to better describe their dynamical behavior and their ability to bind ligands or other macromolecules. This is of fundamental importance for understanding their biological function. In my talk, I will give a survey of methods that we have developed in our laboratory for exploring large amplitude motions of proteins. These methods are based firstly on energy minimizations and molecular dynamics simulations under restraint potentials, and secondly on normal mode techniques. A combination of these methods allows to better take into account the anharmonicity of the energy surface. Particular emphasis will be placed on the question of how global motions may influence the formation of internal cavities in order to better describe ligand fixation or migration. An efficient way to improve today’s drug design strategies may reside in our capacity to describe such large collective motions.

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LECTURE ABTRACTS th

Saturday, 25 August

12:00-12:30

Plenary Lecture 6.2. Tracking protein intramolecular motions in solution with time-resolved Wide Angle X-ray Scattering A. CUPANE 1, M. LEVANTINO 1, M. CAMMARATA 1,2, M. WULFF 2, S. N. AHN 3, H. IHEE 3, F. SCHOTTE 4, AND P. A. ANFINRUD 4 CNISM and Dept. of Physical and Astronomical Sciences, University of Palermo, 90123 Palermo, Italy, [email protected] European Synchrotron Radiation Facility, Grenoble, France. 3 Dept. of Chemistry and School of Molecular Science, KAIST, Daejeon, Republic of Korea. 4 Laboratory of Chemical Physics, NIH, Bethesda, MD 20892-0520, USA. 1 2

Proteins are sequences of aminoacids that fold in 3D objects to accomplish their biological functions. Several studies have shown that protein conformational changes often play a crucial role in function regulation. So far information on molecular motions has been obtained only indirectly through time resolved optical spectroscopy and, more recently, by time resolved protein crystallography. However the use of protein crystals, while allowing model-free atomic resolution reconstruction, may induce stress/strain in the protein and may hinder biological relevant structural changes; moreover, the general problem of allostery cannot be tackled with time resolved crystallography since large scale protein motions are hindered by the constraints imposed by the crystal lattice. By using pulsed x-rays (beamline ID09b at ESRF, Grenoble) to probe the transient protein structures and laser light pulses to excite the protein, we have been able to follow, in solution, the conformational changes of Hemoglobin (Hb) that follow the photolysis of the ligand. The scattering signal was measured in the Q range of 0.1 – 2 Å-1 and in the time interval from ∼ 100ns to ∼10ms; the effect of solution heating due to light absorption was carefully subtracted. Static scattering patterns from deoxy- and carbonmonoxy-Hb were also measured, for comparison. Typical difference signals measured at 178 ns and 31.6 µs from photolysis are reported in the figure; they clearly show that with time-resoved WAXS it is possible, for the first time, to characterize the conformational changes of Hb in solution at both the quaternary and tertiary structural levels. A simple model can account for the time course of the observed difference signals in the whole time window investigated and sheds light on the time sequence of structural events taking place in hemoglobin after ligand photodissociation. Fig. 1. Right: measured difference signal (laser-on – laser-off) after 31.6 µs from photolysis (circles); experimental deoxyHb – carbonmonoxyHb static difference pattern (continuous line); solution difference pattern calculated, using Crysol, from the “R2” and “T” crystallographic structures (dotted line). Left: measured difference signal (laser-on – laser-off) after 178 ns from photolysis (circles); solution difference pattern calculated, using Crysol, from “R2” and “tertiary modified R2” structures .

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LECTURE ABSTRACTS Saturday, 25th August

12:30-13:00

Plenary Lecture 6.3. Monitoring the Subunit Interface and Conformational Dynamics to Unravel the Mechanism of Action of Allosteric Effectors in Human Hemoglobin SCHAY 1, G., SMELLER 1, L., YONETANI 2, T. AND FIDY 1, J. Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, and Research Group for Membrane Biology, HAS, Puskin utca 9 Budapest1088, Hungary 2Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, USA 1

Luminescence and FTIR spectroscopy studies were performed to unravel the way how the local effect of binding allosteric effectors around the central cavity in the HbA tetramer becomes a signal for oxygen binding affinity in the subunit heme pockets. Both the oxy- and deoxy-states of HbA were studied and the effect of binding Cl, DPG, BZF and IHP was investigated. A method has been elaborated by using high hydrostatic pressure to dissociate the HbA tetramers into dimers without affecting the native structure of the subunits, and to determine the Kd of the tetramer->dimer transition based on Trp fluorescence measurements. The data showed that the Kd is affected both in the oxy- and in the deoxy-states of HbA by the binding of allosteric effectors and the extent of the effect followed the order in their efficiency of influencing oxygen affinity. Pressure dependent high resolution fluorescence spectroscopy and temperature dependent phosphorescence lifetime studies based on the emission signal of Zn-protoporphyrin in substituted HbA have been performed to monitor the effect of binding allosteric effectors on the conformational dynamics of the subunits. These results showed that while the overall volume fluctuations do not become significantly affected, the large scale motions around the heme pocket, in the ms range that are related to opening the path for oxygen diffusion become altered by binding the allosteric effectors. These results showed that both the fine structure of subunit interface and the large scale motions of conformational dynamics may play role in forming a signal for oxygen binding from the local structural effect of binding allosteric effectors. Acknowledgements: Support is acknowledged from NIH-FIRCA grant no. TW005924 (T.Y.,J.F.) and from the Hung.Acad.Sci. (J.F., G.Sch.).

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POSTER ABSTRACTS

POSTERS

MACROMOLECULAR STRUCTURE AND FUNCTION

MACROMOLECULAR STRUCTURE AND FUNCTION

1. Searching the autocatalyst in hydrogenase reaction BODÓ 1 G., BRANCA 1, R.M.M., TÓTH 2 Á., HORVÁTH 2 D., BAGYINKA 1, CS 1 2

Institute of Biophysics, Biological Research Center, Temesvári krt. 62., Szeged , H-6726, Hungary Department of Physical Chemistry, University of Szeged, Rerrich ter 1., Szeged, H-6720, Hungary

Hydrogenases are metalloenzymes that catalyze the reaction H2 ↔ 2H+ + 2e- [1-4]. We recently demonstrated that the hydrogenase catalytic reaction includes at least one autocatalytic step [5-7]. This assumption was based on the special patterns of the hydrogenase-uptake reaction in a thin-layer reaction chamber and on the autocatalytic oscillations in the fast absorption kinetics of the methyl viologen-initiated reaction of hydrogenase. The reaction in thin layer allows the determination of the front velocity which can be identified by the intense color of reduced methyl or benzyl viologen. It is still a question what is the autocatalyst of the hydrogenase reaction. In order to obtain an estimation on the nature of the autocatalyst we have determined the dependence of front velocity on the enzyme as well as on substrate (benzyl viologen) concentration. Two different autocatalytic models have been analysed and evaluated, assuming a special form of the enzyme and the reduced substrate as autocatalyst. The comparison of kinetic calculations and the experiments in thin layer suggested the possibility that a special form of the enzyme interacts with the unactivated enzyme forms in order to facilitate the hydrogenase reaction, i.e. a hydrogenase enzyme form is the autocatalyst. Acknowledgement We are grateful for the financial support of the Hungarian Science Foundation [OTKA T049276 and OTKA T049207] and AUTOESKORT Ltd. References [1] Volbeda, A., M.H. Charon, C. Piras, E.C. Hatchikian, M. Frey and J.C. Fontecilla-Camps. 1995. Nature 373: 580587. [2] Armstrong, F. A. 2004. Curr. Opin. Chem. Biol. 8:133–140. [3] Albracht, S. P. J. 2001. Spectroscopy: the functional puzzle.. In Hydrogen as a Fuel: Learning from Nature. R. Cammack, M. Frey, and R. Robson, editors. Taylor and Francis, London, UK; New York, NY. [4] Cammack, R. 2001. Hydrogenases and their activities. In Hydrogen as a Fuel: Learning from Nature. R. Cammack, M. Frey, and R. Robson, editors. Taylor and Francis, London, UK; New York, NY. [5] Bagyinka, C., J. İsz, and S. Száraz. 2003. J. Biol. Chem. 278:20624–20627. [6] İsz, J., and C. Bagyinka. 2005. Biophys. J. 89:1984–1989. [7 ] İsz, J., Bodó, G., Branca, R.M.M., and C. Bagyinka. 2005. Biophys. J. 89:1957–1964.

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2. Quaternary structure dependence of hemoglobin ligand rebinding kinetics under cryogenic conditions LEVANTINO 1, M., CUPANE 1, A. AND FRIEDMAN 2, J.M. Dept. of Physical and Astronomical Sciences, University of Palermo, Via Archirafi 36, Palermo, I-90123, Italy, [email protected] 2 Dept. of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 1

The rebinding reaction of carbon monoxide to myoglobin after flash photolysis has been successfully used in the past as a prototype reaction to yield insights into the energy landscape of proteins [1-2]. Experiments at low temperature have demonstrated that proteins can assume a large number of nearly isoenergetic conformations (conformational substates) and that quantitative information on the properties of the energy landscape can be obtained. In the case of hemoglobin, the same studies are complicated by the presence of the switching between the T and R quaternary conformations, which plays a crucial role in the oxygen transport function of hemoglobin. Here we report time-resolved absorption spectroscopy data at low temperature obtained after photolysis of carbonmonoxy hemoglobin encapsulated in silica gel; sol-gel encapsulation inhibits hemoglobin quaternary relaxation and enables one to study the energy landscape of T state and R state hemoglobin separately [3]. The present data extend the previously reported results [4] in view of the improved time-resolution (5 ns) of the new experimental apparatus employed and the wider temperature interval (20-200 K) investigated. The rebinding process of carbon monoxide to hemoglobin is highly nonexponential in time both for T state and R state hemoglobin, the former being more than one order of magnitude slower, in agreement with data from Austin and coworkers on carp hemoglobin [5]. The analysis of ligand rebinding kinetics reveals large differences between the energy landscapes of T state and R state hemoglobin. At difference from the myoglobin case, it is not possible to fit the data in terms of a temperature independent distribution of enthalpy barriers. The T-R difference, as reflected by the position (first moment) of the enthalpy distributions resulting from our analysis, increases with temperature between 20 and 200 K. The major contribution to this difference originates from a relatively large temperature dependent increase in the enthalpic barrier for the T state species occurring at relatively low temperatures (< 160 K). We speculate that the shift in the enthalpy distribution to T state hemoglobin is likely due to a local rearrangement around the proximal histidine that occurs in response to the initial (nearly instantaneous) out of plane movement of the iron. References [1] Frauenfelder H, Sligar SG, Wolynes PG (1991) Science 254: 1598-1603. [2] Levantino M, Cupane A, Zimanyi L, Ormos P (2004) Proc. Natl. Acad. Sci. USA 101: 14402-14407. [3] Juszczak LJ, Friedman JM (1999) J. Biol. Chem. 274: 30357-30360. [4] Levantino M, Cupane A, Zimanyi L (2003) Biochemistry 42: 4499-4505. [5] Cobau WG, LeGrange JD, Austin RH (1985) Biophys. J. 47: 781-786.

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3. Fluorescence line narrowing and phosphorescence spectroscopy show the importance of global tertiary structural changes in the regulation of hb function. GUSZTÁV SCHAY 1, LÁSZLÓ SMELLER 1, KRISZTIÁN SZIGETI 1, ANDRÁS D. KAPOSI 1, TAKASHI YONETANI 2, JUDIT FIDY 1 1 2

Semmelweis University, Budapest, Hungary, [email protected] Univeristy of Pennsylvania, Philadelphia, PA, USA

The mechanism underlying allostery in hemoglobin is still not completely understood. Various models describing the action of allosteric effectors on hemoglobin function have been published in the literature. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R and T states. In this context, an important question is how far, and how the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl-, DPG, IHP and BZF were bound. We studied oxyHb and a hybrid form (αFeO2)2-(βZn)2 as T state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramerto-dimer transition with characteristic pressures in the 70-200MPa range. The transition is sensitive to the binding of allosteric effectors. We fit the data with a simple model for the tetramer-dimer transition and determine the dissociation constants at atmospheric pressure. In the R state, we observe a stabilizing effect by the allosteric effectors, while in the T-analogue, a stronger destabilizing effect is seen. The order of efficiency is the same in both states, but with opposite trend as: IHP>DPG>Cl-. These results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states. To assess the importance of conformational dynamics in this process we have used Fluorescence line narrowing combined with pressure tuning. These experiments provide information about the change in the isothermal compressibility (which is proportional to the volume fluctuations of the structure) upon the binding of allosteric effectors. The observed changes are relatively small. Phosphorescence lifetime studies indicate that other fluctuations, possibly larger scale motions play more important role in the regulation of Hb function.

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4. De Novo sequencing of a 21-kDa Cytochrome c4 from Thiocapsa roseopersicina by Nano-ESI Ion Trap and FT-ICR Mass Spectrometry R. M. M. BRANCA 1,2 G. BODÓ 1, C. BAGYINKA 1 AND L. PROKAI 2 Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary, [email protected] 2 University of North Texas, Health Science Center, Fort Worth, TX 76107, USA. 1

We had recently discovered a cytochrome c4 in Thiocapsa roseopersicina. This class of cytochromes is usually associated with oxygenic respiratory metabolism, and this was the first cytochrome c4 found in an anaerobic organism. We had already obtained the X-ray diffraction pattern for this protein but cannot map the 3D structure without primary sequence. Because the gene sequence of Thiocapsa roseopersicina has not been determined, our only option was de novo protein sequencing. De novo protein sequencing by mass spectrometry is in its infancy. The “top down” strategy has potential, but has not been a routine method; “bottom up” has been more established, but its success depends on the instrument employed (high resolution/high mass accuracy are preferred, often required). In this presentation, we will share our experience of de novo sequencing of the 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nano-ESI and tandem mass spectrometry. All experiments were performed on a linear ion-trap—Fourier-transform ion-cyclotron resonance (FT-ICR) hybrid instrument (LTQ-FT, Thermo Fischer Scientific, San Jose, Ca, USA). Three different proteolytic enzymes were used to generate digests: trypsin, glutamyl endopeptidase (Glu-C) and the endoproteinase Lys-C (Roche). The proteolytic fragments were separated by on-line gradient reversed-phase micro-liquid chromatography (10 cm x 300 µm i.d. TARGA C18 column, Higgins Analytical, Mountain View, CA, USA) prior to NSI. Fullscan mass spectra were from the FT-ICR, whereas tandem mass spectra (MS/MS) were taken in the ion trap. The software DeNovoX (CSIC-CBMSO, Madrid, Spain, and Thermo Fisher Scientific) was used to generate initial sequence information from MS/MS data. We produced four different sets of proteolytic peptides. Combination of trypsin and Glu-C digestions was used to generate the set with the shortest peptides by cleaving at lysine (K), arginine (R) and glutamic acid (E). The second set was a simple tryptic digest (cleavage at K and R). The third set was a digest by Lys-C, which cleaves at K only. The fourth set used Glu-C digestion (cleavage at E) to enable the linking of sequenced peptides. By this approach, we expected to compensate two major practical sequencing limitations: a) MS/MS spectra of peptides above 1500 Da were too complex to interpret and b) any peptide below 300 Da was usually not detectable. The high mass accuracy afforded by FT-ICR was crucial in solving ambiguities, since it could resolve, for example, the 0.036 mass difference between Q (glutamine) and K. Our presentation will discuss additional practical considerations in detail.

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5. Investigation of cell wall and its components by steady-state fluorescent spectroscopy DJIKANOVIĆ 1, D., RADOTIĆ 1, K.,KALAUZI 1, A. AND JEREMIĆ 2, M. 1Centre

for Multidisciplinary Studies, University of Belgrade, Bul. Despota Stefana 142, 11000 Belgrade, Serbia, [email protected] 2Faculty of Physical Chemistry, University of Belgrade, 11000 Belgrade, Serbia

One of the most important features of plant cells is presence of a cell wall. The plant cell wall serves a variety of functions. The secondary walls of woody tissue and grasses are composed predominantly of cellulose, lignin, and hemicelluloses. The cellulose fibrils are embedded in a network of hemicellulose and lignin. Cellulose is the main constituent of the cell wall. Lignin, as a major structural polymer in the plant cell walls, is the second most abundant polymer on Earth. Lignin is highly branched polymer composed of cross-linked phenyl-propanoid units derived from coniferyl, sinapyl and p-coumaryl alcohols as precursors. It is intertwined and cross-linked with the other macromolecules in the cell walls [1]. Various types of interunit bonds are possible in lignin, leading to different types of substructures [2]. Fluorescence is an intrinsic property of the cell wall and lignin [3]. The structural complexity of lignin makes its fluorescence spectra difficult to interpret. Fluorescence spectroscopy was used as a sensitive analytical tool in the studies of lignin constituents in waters and soils. There were also trials to compare various lignin samples on the basis of deconvolution of their emission spectra. Fluorescence spectroscopy is a sensitive analytical tool in the studies of structure of complex molecular structures. We applied appropriated combination of two mathematical models: symmetric (Gaussian) and (Log-normal) [4]. We analysed emission spectra of the cell walls, corresponding isolated lignin and pure cellulose. Fluorescence spectra were collected using a Fluorolog-3 spectrofluorimeter (Jobin Yvon Horiba, Paris, France) equipped with a 450 W xenon lamp and a photomultiplier tube. We collected 22 spectra for each sample. The spectra were deconvoluted with one Gaussian and appropriated number of Log- normal components. Applied mathematical model shows similarity and differences in fluorophore number among the three samples. As a complementary approach we measured IR spectra of the samples. Combining these two methods it was possible to obtain more precise information about cell wall, cellulose and lignin structure. Acknowledgement Grant 143043 from the Ministry of Science and Technology of the Republic of Serbia supported this study. References [1] Lewis, N. and Yamamoto, E. (1990) Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 455–496. [2] Boerjan, W., Ralph, J. and Baucher, M. (2003) Annu. Rev. Plant Biol. 54, 519–546. [3] Lundquist, K., Josefsson, B. and Nyquist, G. (1978) Holzforschung 32, 27–32. [4] Djikanović, D., Kalauzi, A., Jeremić, M., Mićić, M. And Radotić, K. (2007) Coll. Surf. B: Biointerfaces 54, 188192.

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6. An EPR study of the flexibility of actin filaments reacted with formin proteins TÜNDE KUPI 1, MIKLÓS NYITRAI 1 AND JÓZSEF BELÁGYI 1 1 University

of Pécs, Faculty of Medicine, Department of Biophysics, Szigeti str. 12, Pécs, H-7624, Hungary, [email protected]

Formin proteins are essential in the regulation of the actin cytoskeleton. We used conventional and saturation transfer (ST) electron paramagnetic resonance (EPR) spectroscopy to study the effects of the FH2 domain of a mammalian formin (mDia1) to the rotational motions of actin filaments. Actin filaments from rabbit skeletal muscle were spin labelled with dinitro-fluoro-proxyl on Lys-61 and the labelled actin was reacted with different molar ratio of mDia1-FH2 formin. The results in the conventional EPR time domain showed that formin affected slightly the environment of the labelled sites of actin and induced the decrease of the nanosecond rotational time. Addition of different amount of formin to F-actin produced a significant effect in the microsecond EPR time domain measured by ST-EPR technique. The increase of τ2 with increasing molar ratio of formin to actin is proved to be non-linear. The EPR measurements suggest that the binding of formin to F-actin affected both the segmental and the domain motion of F-actin in the environment of the labelled Lys-61site.

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7. Thermodynamic Characterisation of BeFx.ADP-F-Actin in the Presence of Different Cytotoxins as Revealed by Differential Scanning Calorimetry JÓZSEF ORBÁN, ANDREA VIG, ROLAND KARDOS, BÉLA SOMOGYI†, GÁBOR HILD, MIKLÓS NYITRAI AND DÉNES LİRINCZY University of Pécs, Faculty of Medicine, Department of Biophysics, Pécs, Szigeti str. 12, H-7624, Hungary, [email protected]

Actin is one of the main components of the eukaryote cell matrix and has a central role in various functions in cellular motility. The actin monomer binds an ATP and a divalent ion in its central cleft between its two main domains. The ATP is hydrolized during the polymerization of actin. The final products of hydrolization are an inorganic phosphate (Pi), which dissociates from the actin filaments, and an ADP that remains bound to the protein. Beryllium fluoride (BeFx) is a widely used phosphate analogue and in its complex with ADP mimics the ADP.Pi intermediate step of the ATPase cycle. In our present study we investigated the effect of two toxic peptides, phalloidin and jasplakinolide, on the thermal stability of BeFx.ADP-actin filaments. The thermodynamic properties of different ATP hydrolization states of skeletal F-actin were studied with the method of differential scanning calorimetry (DSC). Addition of BeFx to the actin filaments increased their resistance against heat denaturation. We found an additional increase in the melting temperature when phalloidin was added in equimolar concentration ratio to the actin filaments. This effect was not observed with jasplakinolide. These results provided evidence that although previously the toxins showed similar effects on the actin filaments in many cases, their effect on the thermal stability of BeFx.ADP-actin filaments was substantially different. Based on these observations we concluded that these toxins influence the structure of the actin filaments by using different molecular mechanisms, and the interaction between the toxins and actin is regulated by the binding of nucleotides to actin.

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8. SDSL EPR - GHOST and conformation space modeling for membrane protein structure determination ALEH KAVALENKA 1,2, JANEZ ŠTRANCAR 2, DAVID STOPAR 3, PRIMOŽ ZIHERL 2, VALERIE BELLE 4, MARCUS A. HEMMINGA 5. Belarusian State University, Nezavisimosti Ave 4, Minsk, 220030, Belarus Jožef Stefan Institute, Jamova 39, Ljubljana, 1000, Slovenia, [email protected] 3 University of Ljubljana, Vecvna pot 111 Ljubljana 1000, Slovenia 4 UPR 9036 CNRS, 31 Chemin Joseph-Aiguier, F-13402 MARSEILLE cedex 20, France 5 Wageningen University, Dreijenlaan 3, Wageningen, 6703 HA, Netherlands 1 2

The study of membrane proteins is one of the most important challenges in the field of structural biology. High-resolution X-ray crystallography or solution NMR widely applied for protein structure determination encounters experimental difficulties for studying lipid-embedded protein domains [1,2]. Recent developments of SDLS-EPR data analysis based on EPR spectra simulations, dHEO inverse-problem solving, GHOST condensation enables us to obtain the local structural information of a specific site of the protein. Multiple SDSL-EPR enhanced by simulation of local rotational conformation spaces (SICS) becomes a powerful tool to tackle the structural properties of the membrane proteins. Although this approach is being developed for lipid-embedded protein domains its capabilities may be also extended to structural characterizations of such systems as very flexible water soluble proteins. Here we present the basics of conformation spaces simulations (fig. 1) and draw its potentials for the interpretation of multiple SDSL-EPR experimental data to the membrane protein structure determination. Unrestricted conformational space

B

Restrictions by backbone

Restrictions by neighborhood

H

F

D

Restrictions by lipids

χ χ χ

χ

more restricted

A

C

E

G

less restricted

Fig. 1. Conformational space modeling and restrictions calculation. A, Single bonds free rotations of the spin label sidechain. B, Unrestricted spin label rotation conformational space (CS). C, Steric overlap of the spin label CS with the protein backbone. D, The restriction of the spin label CS due to the steric overlap with the backbone. E, Spin label shares the space with the neighbouring amino acids side-chains. F, Spin label CS restrictions including CS overlaps with the neighbouring amino acids side-chains. G, Lipids acyl chains highly restrict those conformations that are perpendicular to the membrane normal. H, The final spin CS of the spin label subject to all three types of restrictions. References [1] Torres, J, Stevens, TJ, Samso, M (2003) Trends in biochemical sciences 28(3): 137-144. [2] Hemminga, MA. (2007) In: ESR Spectroscopy in Membrane Biophysics (Hemminga MA, Berliner L, eds.), Springer, pp 1-16.

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9. The effect of the actin binding proteins on the conformation of the ATP binding cleft on actin ROLAND KARDOS 1, KINGA POZSONYI 1, ANDREA VIG 1, MIKLÓS NYITRAI 1, ELISA NEVALAINEN 2, PEKKA LAPPALAINEN 2 AND GÁBOR HILD 1 1University 2Program

of Pécs, Faculty of Medicine, Department of Biophysics, Szigeti str. 12, Pécs, H-7624, Hungary, [email protected] in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland

Actin is one of the most abundant proteins in all eukaryotic cells. Both the monomer and the polymer form of the actin can be found in the cells under physiological conditions. The dynamics and organization of the actin filaments in cells are regulated by a large amount of actin-binding proteins. Cofilin is a conserved regulator of cytoskeletal dynamics that promotes depolymerization of actin filaments and can inhibit the nucleotide exchange on actin monomers as well. Another regulator of the intracellular actin monomer pool is profilin, which facilitates the incorporation of actin monomers to the barbed ends of the filaments. Most profilins also promote the nucleotide exchange on actin monomers. We investigated the effect of cofilin and profilin on the structure of actin monomers around the ATP binding pocket. The fluorescence of the actin bound etheno-ATP (ε-ATP) was quenched with a neutral quencher (acrylamide). The data were analysed with a modified form of the Stern-Volmer equation. With this special form of the equation it is possible to separate the fluorescence signal coming from the actin bound and the free ε-ATP in the solution. The experiments revealed that in the presence of cofilin the accessibility of the bound ε-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the presence of cofilin. Contrary to this, in the presence of profilin the accessibility of the bound ε-ATP increased, indicating a more approachable protein matrix around the ATP-binding pocket. This more open structure may be actively involved in the nucleotide exchange facilitated by the profilin molecule.

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10. Thermal Stability of Thrombin Binding Aptamers in the Presence of Stabilizing Cations PONIKOVA 1, S., ANTALIK 2,3, M. AND HIANIK 1, T. Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska dolina F1, Bratislava, 842 48, Slovakia, [email protected] 2 Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, Kosice, 040 01, Slovakia 3 Faculty of Science, P.J. Safarik University, Moyzesova 11, Kosice, 041 54, Slovakia 1

Thrombin binding DNA aptamers are single stranded oligonucleotides that specifically bind to human αthrombin. First aptamer competes with fibrin for binding to fibrinogen-recognition exosite, whereas second binds to heparin-binding exosite. Two DNA aptamers directed against two separate exosites on human αthrombin differs in nucleotide sequences, but in certain conditions both forms an unimolecular quadruplex structure. In this work we used circular dichroism (CD) to determine spectral and thermodynamic characteristics of the formation of G-quadruplexes in both thrombin binding aptamers. We showed, that aptamers forms compact tertraplex structure with antiparallel strand orientation in the presence of potassium cations. This was readily identified through characteristic CD spectra of antiparallel quadruplex conformation. We also investigated CD spectral changes at various temperatures. At low temperature conditions without potassium we observed similar CD spectra as with stabilizing potassium cations. The intensity of CD bands at low temperature was lower than in the presence of potassium. This suggests that aptamers form quadruplex structure but it is not well-ordered as in the presence of stabilizing cations. With increase of the temperature the intensity of CD bands decreases for both aptamers. We also examined chnges in melting temperatures and vant’Hoff enthalpies as a function of salt concentration. The measurements showed that with increasing of ionic strength the melting temperature increases and changes of vant’Hoff enthalpy take place. The obtained data allowed us to determine changes in free energy ∆G and heat capacity ∆Cp. the values of these parameters were different for each fibrinogen-recognition exosite specific aptamer and heparin-binding exosite specific aptamer. Acknowledgement The authors are grateful to the Agency for Promotion Research and Development (Contracts No. APVT-20-P01705), to VEGA Grant agency (Projects No. 1/4016/07 and No. 2/6167/06) and to Comenius University for their financial support (UK /368/2007).

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11. Disaggregation effect of Acridines BELLOVA 1, A., GAZOVA 2, Z., IMRICH 1, J., KRISTIAN 1 P., DAXNEROVA 1 Z. AND ANTALIK 1,2 M. 1 2

Faculty of Science, P. J. Safarik University, Moyzesova 11, 041 54 Kosice, Slovakia, [email protected] Department of Biophysics, Institute of Experimental Physics, Watsonova 47, 040 01Kosice, Slovakia

A range of neurodegenerative and fatal human diseases, including Alzheimer´s disease, Creuzfeld-Jakob disease, diabetes type II, is associated with the deposition of proteins in human organs [1]. The protein deposits consist of amyloid fibrils formed by a single predominant protein that is characteristic of each disease. Lysozyme deposits are characteristic of systemic amyloidoses and can be found in various part of body. A promising strategy to achieve prevention of protein deposition diseases is identification of compounds which are able to inhibit protein polymerization or disaggregate protein deposits [2]. Acridines are a special class of organic compounds due to their possible utility as cancer drugs [3]. The ability of acridines to be effective also in the case of protein deposit diseases has been investigated. We screened a small library of structurally distinct acridine derivatives (25) with various bulky side groups for their ability to inhibit lysozyme fibril formation in vitro. By Thioflavin S fluorescence the percentage of lysozyme aggregation were detected in presence of Fig. 1. Inhibition of lysozyme fibril formation ( ) and disassembly of pre-formed fibrils ( ) induced by compounds. We found that structure of the acridine acridine derivatives side chains effects the antiaggregation activity. There is a group of acridine derivatives able to inhibit lysozyme fibril formation or destroy pre-formed fibrils at low micromolar concentrations (IC50 and DC50 values between 5-50 µM). These abilities were confirmed also by electron microscopy and Congo red assay. Compounds belong to spiroacridines and tetrahydro-acridines have no disaggregation effect. Moreover, the lysozyme fibril growth was accelerated in presence of some tetrahydro-acridines (Fig.1). We found out that disaggregation activity of acridines is affected by planar template and by the composition and structure of side chains. Acknowledgement This work was supported by the research grants from the Slovak Grant Agency VEGA no. 7055, 2471, 6167 and VVGS grant PF8/2007/CH. References [1] Buccianti, M, Giannoni, E, Chiti, F, Baroni, F, Formigli, L, Zurdo, J, Taddei, N, Ramponi, G, Dobson CM, Stefani, M (2002) Nature 416: 507-511. [2] Pickhardt, M, Gazova, Z, von Bergen, M, Khlistounova, I, Wang Y-P, Hascher, A, Mandelkow, E-M, Biernat, J, Mandelkow, E (2005) J Biol Chem 280: 3628-3635. [3] Hansen, JB, Langvad, E, Frandsen, F, Buchardt, O (1983) J Med Chem 26: 1510-1514.

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12. Effects of different salts on the catalytic activity and structure of membrane bound hydrogenase from purple photosynthetic bacteria Thiocapsa

roseopersicina BODÓ G., BRANCA, R.M.M. AND BAGYINKA, CS. Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62., Szeged, 6726, Hungary, [email protected]

Hydrogenases are metalloenzymes which catalyse the reversible reduction and oxidation of molecular hydrogen and are present mostly in prokaryotes and in some eukaryotes [1]. The catalytic mechanism was investigated mainly by different spectroscopic methods (electron paramagnetic resonance, infrared spectroscopy, etc.), some models were established, but the whole process is not well explained yet. The membrane bound hydrogenase from purple photosynthetic bacteria, Thiocapsa roseopersicina (HynSL) has high stability against oxygen, heat and proteolytic digestion which properties hydrogenases do not have in general [2]. Several enzyme forms could be distinguished by spectroscopic methods [3] and under certain conditions different conformers could also be seen by two dimensional SDS (sodium-dodecylsulphate) gelelectroforesis [4]. Our previous results demonstrate an autocatalytic mechanism during the reaction cycle of hydrogenase [5,6]. This assumption was based on the special patterns of the hydrogenaseuptake reaction in a thin-layer reaction chamber and on the autocatalytic oscillations in the fast absorption kinetics of the methyl viologen-initiated reaction of hydrogenase. The assumption of an autocatalytic step explains most of the contradictory findings in previous publications. Salts, in the concentration range of 0.01 M - 1 M, are known to affect the properties of proteins in solution, including stability, enzyme activity, etc. [7]. Such effects follow a trend called Hofmeister series [8]. Hofmeister salt effect on enzyme activity can be explained by induced conformational changes and stabilization of a conformer of the protein [9]. The spatial pattern of hydrogen oxidizing reaction studied in thin layer reaction chamber changed dramatically when chaotropic salts were present in the medium, while there was no change in the case of cosmotropic salts. Our assumption is that during the reaction of hydrogenase a protein conformation change happens and this process could be affected by different salts. We have also examined the hydrogen oxidizing and reducing activity of hydrogenase in the presence of different salts of Hofmeister series. The structural changes in hydrogenase, induced by different salts were also measured by different spectroscopic methods (circular dichroism, infrared, absorption). Acknowledgement We are grateful for the financial support of the Hungarian Science Foundation [OTKA T049276 and OTKA T049207] and AUTOESKORT Ltd. RMMB acknowledges the financial support of the Portuguese Science and Technology Foundation under the PhD fellowship of POCTI, SFRH/BD/13128/2003. References [1] Vignais, PM, Billoud, B, Meyer J (2001) FEMS Microbiol Rev 25(4): 455-501. [2] Kovacs, KL, Bagyinka, Cs, Tigyi, G (1988) BBA 935:166-72. [3] Cammack, R, Bagyinka, Cs, Kovacs, KL (1989) Eur J Biochem 182:357-62. [4] Tigyi, G, Bagyinka, Cs, Kovacs, KL (1986) Biochimie 68:69-74. [5] İsz, J, Bagyinka, Cs (2005) Biophys J 89(3):1984-9. [6] İsz, J, Bodo, G, Branca, RMM, Bagyinka, Cs (2005) Biophys J 89(3):1957-64. [7] Cacace, MG, Landau, EM, Ramsden, JJ (1997) Q Rev Biophys 30(3):241-77. [8] Kunz, W, Henle, J, Ninham, BW (2004) Curr Opin Colloid Interface Sci 9:19-37. [9] Der, A, Kelemen, L, Fabian, L, Taneva, SG, Fodor, E, Pali, T, Cupane, A, Cacace, MG, Ramsden, JJ (2007) J Phys Chem B in press

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13. Hierarchic finite level energy landscape model - to describe the refolding kinetics of phosphoglycerate kinase OSVÁTH 1, S., HERÉNYI 1, L., ZÁVODSZKY 2, P., KÖHLER 3, G., AND FIDY 1, J. Dept. of Biophysics and Radiation Biology, Semmelweis University, Puskin 9, Budapest, H-1088, Hungary, [email protected] Institute of Enzymology, Biological Research Center of Szeged, Karolina 29; Budapest, H-1518, Hungary 3 Institute of Theoretical Chemistry and Structural Biology, University of Vienna, Rennweg 95B, Vienna, A-1090, Austria 1 2

One of the most intriguing predictions of energy landscape models is the existence of non-exponential protein folding kinetics caused by hierarchical structures in the landscapes. Here we present a hierarchic landscape model to describe folding of yeast phosphoglycerate kinase. Refolding was initiated from the guanidine-unfolded state by stopped-flow or manual mixing, and monitored by tryptophan fluorescence from 1 ms to 15 minutes. The strategy to build a model that describes folding of yeast phosphoglycerate kinase was to start from the simplest paradigm and modify it stepwise to the necessary minimal extent after repeated comparisons with the experiments. We made no a priori assumptions about the folding landscape. The result was a Hierarchic Finite Level landscape model that quantitatively describes the refolding of yeast phosphoglycerate kinase. The early steps of the folding process happen in the upper region of the landscape, where the surface has a hierarchic structure. This leads to stretched kinetics in the early phase of the folding. The lower region of the energy landscape is dominated by a trap that reflects the accumulation of molten globule intermediate state. From this intermediate, the protein can reach the global energy minimum corresponding to the native state through a cross-barrier folding step.

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14. Heme pocket dynamics of carbonmonoxyheme proteins: Infrared absorption study of horseradish peroxidase KAPOSI 1, A.D., VANDERKOOI 2, J.M. AND STAVROV 3, S.S. Dept. of Biophysics/Radiation Biology, Semmelweis University, Puskin u. 9, Budapest H-1088, Hungary, [email protected] Johnson Research Foundation, Dept. of Biochemistry/Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 3 Sackler Institute of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, P.O.B.39040, Tel Aviv 69978, Israel 1 2

Heme proteins (e .g. horseradish peroxidase, HRP) significantly change configuration upon coordination of different ligands [1]. The temperature dependences of the infrared absorption CO bands of carboxy complexes of HRP in glycerol/water mixture at pH 6.0 and 9.3 are interpreted using the theory of optical absorption bandshape [2, 3] (Fig.1). The bands’ anharmonic behavior is explained assuming that there is higher-energy set of conformational substates (CSSh), which are populated upon heating and correspond to the protein substates with disordered water molecule in the heme pocket. Analysis of the second moments of the CO bands of the carboxy complexes of myoglobin (Mb(CO)) and hemoglobin (Hb(CO)), and of HRP(CO) with benzohydroxamic acid (HRP(CO)+BHA) shows that the low energy CSSh exists also in the “open” conformation of Mb(CO), where the heme pocket is spacious enough to accommodate a water molecule. In the Fig. 1. 1.Temperature dependence of the CO HRP(CO)+BHA and “closed” conformations of Mb(CO) infrared absorption band of HRP(CO) in and Hb(CO) the heme pocket is packed with BHA and glycerol/water solvent at pH 6.0 (a) and pH different amino acids, the CSSh has much higher energy and 9.3 (b). The spectra from top to bottom is hardly populated even at the highest temperatures. correspond to the temperatures 12, 50, 90, 130, 170, 200, 230, 260 and 290 K. Therefore only motions of these amino acids contribute to the band broadening. These motions are linked to the protein surface and frozen in the glassy matrix, whereas in the liquid solvent they are harmonic. Thus the second moment of the CO band is temperature independent in glass and is proportional to the temperature in liquid. The temperature dependence of the second moment of the CO peak of HRP(CO) in the trehalose glass exhibits linear coupling to an oscillator. This harmonic behavior of the central CO peak in the infrared absorption spectra of HRP(CO) in trehalose manifests electrostatic coupling of the CO vibration to an oscillator which is disengaged from the protein surface motion. We speculate that this oscillator is a trehalose molecule located in the heme pocket of HRP [3]. References [1] Ansari A, Berendzen J, Braunstein D, Cowen BR, Frauenfelder H, Hong MK, Iben IET, Johnson JB, Ormos P, Sauke TB, Scholl R, Schulte A, Steinbach PJ, Vittitow J, Young RD (1987) Biophys Chem 26:337-355. [2] Stavrov SS, Wright WW, Vanderkooi JM, Fidy J, Kaposi AD (2002) Biopolymers 67:255-258 [3] Kaposi AD, Vanderkooi JM, Stavrov SS (2006) Biophys J 91: 4191-4200.

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15. In vivo EPR detection of spin-labelled ovalbumin applied subcutaneously in mice ABRAMOVIC 1, Z., BRGLES 2, M., HABJANEC 2, L., FRKANEC 2, R., TOMASIC 2, J. AND SENTJURC 1, M. 1 2

Jozef Stefan Institute, Laboratory of Biophysics, Jamova 39, Ljubljana, 1000, Slovenia, [email protected] Institute of Immunology Inc.,Rockefellerova 10, Zagreb, 10001, Croatia

I/I0

Ovalbumin (OVA) is a glycoprotein often used as a model antigen of low immunogenicity in assessment of novel adjuvants. Liposomes are phospholipid vesicles, which are used as drug delivery systems and are known as immunological adjuvants. In our previous studies we have investigated effect of liposomes with OVA as antigen on the immune reaction in mice1. Interaction of OVA with liposom bilayers was also studied using EPR spectroscopy2. The aim of this study was to investigate our model antigen OVA as well as OVA entrapped in liposomes in vivo and ex vivo by electron paramagnetic resonance (EPR) spectroscopy in order to establish the distribution and pharmacokinetics of OVA (free or entrapped) within the body after subcutaneous (s.c.) application in mice. For this purpose the OVA was labelled with 4-maleimido-2,2,6,6tetramethylpiperidine-1-oxyl (TEMPO-maleimide), covalently attached to the SH group of cystein within the protein. First we have applied spin labelled OVA (SLOVA) subcutaneously to mice and monitored the 1.0 kinetics of EPR spectra intensity decrease of SLOVA at the site of application using low frequency 0.8 EPR. Intensity of EPR signal decreased with time in linear and reproducible manner. Approximately 50 0.6 min after application of SL-OVA its EPR signal diminished for 50 %. To investigate whether the 0.4 signal decrease was due to the reduction of spin labels by oxy-redoxy systems in the in vivo environment or because of the protein clearance by 0.2 SL-OVA blood flow we applied s.c. SL-OVA together with SL-OVA + K3(FeCN)6 the oxidizig agent K3(FeCN)6 (5 mM), which 0.0 oxidizes hydroxylamines back to nitroxide. Results 0 10 20 30 40 50 showed that EPR spectra intensity decay was slower t (min) with K3(FeCN)6 and intensity diminished only for approximately 30 % within 50 minutes. The results Fig. 1. EPR spectra intensity decay curves of SL-OVA indicate that nitroxide group on SL-OVA is partially and SL-OVA with K3(FeCN)6 within the skin of reduced at the site of application, however mice (I0 – EPR intensity immediately after SLapproximately 30 per cent signal decrease is due to OVA application, I – EPR intensity at certain time the clearance (Fig. 1). point). For ex vivo experiments skin, spleen and liver were dissected and frozen in liquid nitrogen one hour after application of SL-OVA, (free or encapsulated) and signals measured afterwards on X-band spectrometer at 130 K. EPR spectra intensity from skin treated with both free or encapsulated SL-OVA is much higher from the control (skin of untreated mice). Also the EPR spectra intensity of treated liver was higher than the control, while the spectra of treated spleen were the same as in the control samples. This indicates that one hour after s.c. application, SL-OVA (free or entrapped in liposomes) is distributed into the liver but not into the spleen. References [1] Habjanec L, Frkanec R, Halassy B, Tomasic J (2006) J. Liposome Res. 16(1): 1-16. [2] Brgles M, Mirosavljevic K, Noethig-Laslo V, Frkanec R, Tomasic J (2007) Int. J. Biol. Macromol. 40(4): 312-318.

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16. Changes in Raman spectra of high density lipoprotein induced by PRODAN embedment KRILOV, D., BALARIN, M. AND BRNJAS-KRALJEVIĆ, J. Department of Physics and Biophysics, University of Zagreb, School of Medicine, Šalata 3, Zagreb, 10 000, Croatia, [email protected]

It is widely accepted that high density lipoproteins (HDL) have the protective role in the development of atherosclerosis, mainly by reversible cholesterol transport. It is maintained by the ability of HDL to dissolve apolar molecules and to transport them successfully through the water environment. Such external compounds induce conformational changes, which could affect usual metabolic pathway of HDL particle, specifically its ability to bind to the receptors at the cell membrane. The aim of this preliminary study was to investigate if the vibrational modes observed in Raman spectra of intact HDL particles are sensitive to the conformational changes induced by the embedment of a simple apolar external compound, PRODAN, into the particle. Due to its ampiphatic nature, we expected that the PRODAN molecule should reside at the interface between apolipoprotein belt and lipid moiety. The study started with the elaboration of the Raman spectral analysis of native HDL in the whole frequency region. The complexity of the Raman spectrum of HDL is the consequence of large number of individual vibrational modes from lipids and amino acids including protein amide I band. Therefore, the second derivative spectra were needed to resolve the individual lines. For the assignment of the lines, the comparison with the data from the literature was used. In order to distinguish which particular vibration modes are from lipids and which from amino acids in some frequency regions where they overlap, the liposomes, composed from lipids which are present in HDL were prepared and their Raman spectra were recorded. The spectra from native HDL and the particles with PRODAN were compared in three distinct frequency regions: high frequency region with the modes from end groups belonging to the lipid “tails” and amino acid side chains, fingerprint region and low frequency region where mostly skeletal modes are present. The influence of PRODAN is observed in all three frequency regions: in high frequency region, as the shift of some end group lines to lower frequencies; in fingerprint region, as the increasing of the contribution of beta strand/beta turn structure and decreasing of disorder structure in the protein amide I band and in the low frequency region, as the higher intensity of skeletal modes and structural change of Tyrosine Fermi doublet, which is known to be very sensitive to the molecular surrounding. On the basis of these observations we propose that PRODAN molecule is located between terminal parts of lipid chains and has an active role on the dynamics of apolar and basic amino acids in its vicinity. Its influence results also in the change of the secondary structure of beta domains of apolipoprotein. The further analysis of separated lipid and protein fractions of HDL will help in better and more complete understanding of interaction of PRODAN with HDL. Acknowledgement We acknowledge Mrs. Branka Dejanović from Institute Ruñer Bošković for the help with HDL preparation. We also thank Ministry of Science, Education and Sports for financial support, grants No. 0108043 and 3105.

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17. Efficient modeling of polysaccharide conformations based on SAXS experiments I. DOGSA 1, J. ŠTRANCAR 1, D. STOPAR 2. 1”Jožef Stefan” Institute, Department for Condensed Matter Physics, Jamova 39, Ljubljana SI-1000, Slovenia, [email protected] 2 University of Ljubljana, Biotechnical Faculty, Laboratory of Microbiology, Večna pot 111, Ljubljana SI-1000, Slovenia

The determination of polysaccharide structures poses a significant challenge in structural biology of macromolecules. The method of choice is Small Angle X-Ray scattering (SAXS), which can reveal structural information on the 1-100 nm scale, typically inaccessible to other techniques. In this study we have developed a new string of beads model, capable of fitting SAXS curves of polysaccharides of various complexity. The model automatically scans through different polysaccharide single helices with different pitch, number of monomers per pitch, and cross-sectional radius, as well as random coils with exclusion volume taken into an account. The model generates an extensive set of polysaccharide conformations and calculates corresponding SAXS scattering curves, which are compared to SAXS experimental curves. All conformations with root mean scaled standard deviation (RMSSD) ≤1 are retained as the solutions. The model was tested on pullulan oligomers, gellan gum, and isolated bacterial EPS. To account for electrostatic repulsion in gellan gum the Gaussian repulsion interaction term was used. In isolated bacterial EPS Debye heterogeneities were used to resolve the structure. The structural properties of the population of solutions were consistent with spectroscopic and thermodynamical data of pullulan oligomeres and gellan gum. The model is capable of providing a solution without a priori knowledge of the polysaccharide structure. the email address of the presenting author only.

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18. Marine Gel Network MIŠIĆ 1, T., SVETLIČIĆ 1, V., ŽUTIĆ 1 V., JENKO 2, M., MANDRINO 2, D., GODES 2, M. AND MILUN 3, M. Rudjer Boskovic Institute, Bijenicka 54, Zagreb, 10000, Croatia, [email protected] of Metals and Technology, Lepi pot 11, Ljubljana, 1001, Slovenia 3 Insitute of Physics, Bijenicka 46, Zagreb, 10000, Croatia 1

2 Insitute

The macroscopic marine gel phase appears episodically in the northern Adriatic Sea. The phenomenon manifests itself in rapid production of enormous amounts of gelatinous matter in the water column and on the sea surface. Current views leave no doubt on phytoplankton as a proximal source of polymers constituting the gel network, but the mechanism leading to its rapid formation remains unknown. The mechanism of marine gel formation, its stability and the role of gel state in marine ecosystem is becoming a most challenging exercise in converging disciplines of marine chemistry, microbiology and biophysics. [1] Atomic force microscopy (AFM) imaging of native marine gel demonstrates that an important fraction of specimen consists of entangled fibrils (Figure 1). The gel structure exhibits a continuous network of entangled monomolecular polymeric fibrils with solvent cavities ranging from 150 to 500 nm. There is strong evidence that the fibrils are mainly polysaccharides since chemical analysis of marine gel composition reveled sulfonated and carboxylated polysaccharides as main constituents[2]. Site specific elemantal analysis (SEM/EDS, AES, XPS) clearly showed specific sites of accumulation of silica within gel network. References [1] Žutić, V, Svetličić, V, (2000) In: The Handbook of Environmental Chemistry, Vol. 5, Part D, Marine Chemistry (Wangersky P, ed.), Springer-Verlag, Berlin-Heidelberg, pp. 150-165. [2] Magaletti, E, Urbani, R, Sist, P, Ferrari, CR, Cicero, AM (2004) Eur. J. Phycol., 39 (2) : 133-142.

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(3µm x 3µm x 10nm) Fig. 1. AFM height image of marine gel on mica in air

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19. Linear and Nonlinear Compressibility of the Enzyme Horseradish Peroxidase SUISALU 1, A., KUZNETSOV 1, A., MAURING 1, K., KIKAS 1, J., HERÉNYI 2, L. AND FIDY 2, J. 1 2

Institute of Physics, University of Tartu, Riia 142, Tartu, EE2400, Estonia Department of Biophysics and Radiation Biology, Semmelweis University, POB. 263, Budapest, H-1444, Hungary, [email protected]

High resolution, total luminescence spectroscopy (TLS) was used to study the effect of high pressure through the prostetic group in horseradish peroxidase (HRP), which was free-base mesoporphyrin IX (MP) substituting the heme of the enzyme. Series of fluorescence line-narrowing (FLN) spectra were registered at 5 K to determine the (0,0) bands shape. Specific diagonals of the two dimensional array of data give the inhomogenous distribution function (IDF), in which at least three bands of the respective tautomeric forms are well distinguishable (B1, B2, B3). In the range of 0–440 MPa pressure-induced repopulation, red-shift and broadening of the bands were observable. Based on the fitted parameters of IDF-s, isothermal compressibilities of MP-HRP at different tautomeric states were determined. These values in B1 and B2 bands are 0.06 GPa–1 and 0.21 GPa–1 respectively, which are smaller than that found earlier as 0.11 GPa–1 and 0.29 GPa–1 by fine-tuning the pressure in the range up to 2 MPa. In B3 band the compressibility is 0.30 GPa–1, the same value what was measured earlier in the lower pressure range. Thus we have to critically review the widely accepted statement: mechanically, the protein molecule is a highly nonlinear system.

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20. Determination of native and denatured BSA by chronopotentiometric peak H V. OSTATNÁ AND E. PALEČEK Institute of Biophysics, Academy of Sciences of the Czech Republic v.v.i., Kralovopolska 135, 612 65 Brno, Czech Republic, [email protected]

Proteins structure can be changed by denaturation [1]. Heating, adding a chemical denaturant such as guanidinium chloride (GdmCl) or urea and changing pH are commonly applied. Studies of protein denaturation by means of different methods have been important for better understanding thermodynamic stability and other properties of individual proteins.Proteomics and biomedicine are booming scientific fields rapidly acquiring new methods of protein analysis. Fast techniques applicable for preliminary tests of the protein properties and structural changes are sought. Methods of electrochemical analysis have been little utilized in these fields. Earlier d.c. polarographic data showed similar, albeit much smaller differences between native and denatured proteins using the so-called Brdicka catalytic reaction obtained in the presence of cobalt ions [2,3]. Such differences were recently observed with native and denatured BSA using d.c. polarography (with DME) while d.c. voltammetry (with HMDE) of the same samples was not able to discriminate native from denatured BSA[4]. Peptides and proteins produce at mercury electrodes a chronopotentiometric peak at highly negative potentials (peak H), which is due to the catalytic hydrogen evolution. This peak differs from the previously described polarographic and voltammetric electrocatalytic signals of proteins [5] (i) by its ability to detect peptides and proteins down to nanomolar and subnanomolar concentrations and (ii) by its remarkable sensitivity to local and global changes in protein structures. We studied native and denatured BSA by means of constant current chronopotentiometric stripping analysis in borate buffer. Our results showed that both urea-denatured and GdmCl-denatured BSA produced substantially higher peaks H than native BSA samples measured under the same conditions. In difference to the effect of urea, the presence of non-denaturing concentrations of GdmCl in the sample greatly enhanced peak H both in native and in denatured BSA. When denaturing disulfide bonds-containing proteins these have to be reduced to obtain fully Figure Peak H of 100 nM unfolded protein. We reduced BSA using tris (2-carboxyl-ethyl) phosphine native (solid line) and hydrochloride, i.e. the agent frequently used for protein disulfide group denatured BSA (bold line). reduction. We used peak H to follow kinetics of BSA aggregation, 0.15 M borate buffer, pH 9.7 with 70 mM GdmCl was used denaturation and/or reduction. Qualitatively similar results were obtained also with other proteins in as a background electrolyte. native and denatured states, such as human serum albumin, γ- and αglobulin. CPS peak H was also obtained with mercury meniscus-modified silver solid amalgam electrode. Acknowledgement Skillful technical assistance of Mrs. Lida Rimankova. This work was supported from grants the Grant Agency of the Czech Republic 301/07/0490, 202/07/P497 and Ministry of Education, Youth and Sports, CR, LC06035. References [1] C. N. Pace, G. R. Grimsley, J. M. Scholtz, in: J. Buchner, T. Kiefhaber (Eds.). Protein folding handbook, vol.1, Wiley-VCH, Weinheim, 2005: p. 45. [2] M. Brezina, P. Zuman, Polarography in medicine, biochemistry and pharmacy. Interscience, New York, 1958. [3] P. Zuman, E. Palecek, in: E. Palecek, F. Scheller, J. Wang (Eds.). Electrochemistry of nucleic acids and proteins. Towards electrochemical sensors for genomics and proteomics., 2005: p.755. [4] V. Ostatna, B. Dogan, B. Uslu, S. Ozkan, E. Palecek, J. Electroanal. Chem. 593: 172-178. [5] E. Palecek, as ref.3: pp. 690-750.

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21. FRET, LSC and MD-modeling reveals the conformation of the C-terminal segments of the c-Jun-mRFP1 and Fos-EGFP heterodimer N. SZALÓKI 1, N. BAUDENDISTEL 3, C-W. VON DER LIETH 3, M. DOAN 2, S. DAMJANOVICH 1, J. LANGOWSKI 3, ZS. BACSÓ 2, K. TÓTH 3, G. VÁMOSI 1 1Cell

Biology and Signaling Research Group of the Hung. Acad. Sci. and 2Dept. of Biophysics and Cell Biology, Medical and Health Science Center, Univ. of Debrecen, Hungary, [email protected] 3Div. Biophysics of Macromolecules, DKFZ, Heidelberg, Germany

Fos and Jun proteins belong to the AP-1 (activator protein 1) family of transcription factors; they act in a dimer form. They are members of the basic-region leucine-zipper (bZIP) proteins and bind to palindromic AP-1 sites. We have previously shown that the fluorescent fusion proteins Fos-EGFP and Jun-mRFP1, cotransfected in HeLa cells, formed stable complexes [1]. Crystallographic data are only available for the DNA binding and dimerization domains of the complex; there is no structural information on the C terminal transactivation domains. Here we studied the relative position of the C terminal domains by fluorescence resonance energy transfer measured by flow cytometry, confocal microscopy and laser scanning cytometry. We developed an iterative evaluation method to exactly determine the quantitative ratio of the donor (GFP or CFP) to the acceptor (mRFP1 or YFP), as well as the FRET efficiency. The full length Jun-GFP and Fos-mRFP1 dimers displayed a FRET efficiency of E=4±1%, corresponding to a distance of ~8±1 nm when measured by flow cytometry. Deletion of the protruding 165 AA at the C terminus of Fos brought the dyes closer together and resulted in E=11±1% (~6.7±0.2 nm between the labels). The EGFP-mRFP1 fusion protein (pos. control) yielded a mean FRET efficiency of ~27.7%, while the independently expressed GFP and mRFP1 (neg. control) resulted in ~0.9%. With increasing donor-acceptor ratio the FRET efficiency was increased as expected. Similar results were obtained by CLSM. Laser Scanning Cytometer (LSC) is a microscope-based cytofluorometer, by which fluorescence data and light scatter of several thousand cells can be measured in a few minutes; meanwhile we get morphological information from each cell. Repeating the measurements with the shortened Fos215-CFP + Jun-YFP pair by using LSC we got similar results: the FRET efficiency was ~7.7±4%. The positive control (CFP-YFP) resulted in E=28±2.5%, while we got 0.7±0.5% for the negative control. These results validate LSC as an alternative method for FRET determinations using the CFP-YFP pair. Molecular dynamic simulations predict a dye-to-dye distance of 6.7±0.2 nm for the dimer between JunmRFP1 and the shortened Fos-EGFP, in good agreement with our FRET data. A wide variety of models could be developed for the full length dimer, with possible dye-to-dye distances varying largely between 6 and 20 nm. From our FRET results we can conclude that more than half of the occurring dye-to-dye distances are between 6 and 10 nm. Combining FRET with molecular dynamic modeling can distinguish this way between theoretically possible conformations to yield a clearer and more detailed picture of proteinprotein interactions in live cells. Acknowledgement This research was supported by the following grants: OTKA T48745, NK61412, ETT 070/2006, 065/2006, MÖBDAAD 2006/34. References [1] N. Baudendistel, W. Waldeck, J. Langowski (2005) Two-Hybrid Fluorescence Cross-Correlation Spectroscopy Detects Protein-Protein Interactions In Vivo, ChemPhysChem 6: 984-990.

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22. Heterologous overexpression of eukaryotic cytochrome c and cytochrome c heme lyase in Escherichia coli for biophysical studies TENGER 1, K., KHOROSHYY 1, P., KOVÁCS 1,2, K.L., RÁKHELY 1,2, G. AND ZIMÁNYI 1, L. Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62., Szeged, H-6701, Hungary, [email protected] 2 Department of Biotechnology, University of Szeged, P.O.Box 521, Szeged, H-6701, Hungary, 1

We improved an already existing cytochrome c expression system to a reliable, tightly controllable one to achieve a higher expression yield for single cysteine mutants of horse cytochrome c [1]. The protein is heterologously overexpressed in E. coli together with the maturation coordinating enzyme heme lyase from yeast. Various plasmid constructs and host strains were tested for protein expression yield and routinely around 35 mg/L yield was achieved, which is a good result for a posttranslationally modified enzyme. The purpose of producing cysteine mutants is to position accessible cysteine residues on the surface of cytochrome c which can be labeled with a photoactive redox dye for intramolecular and intermolecular electron transfer measurements [2]. The system is also used in a general investigation of posttranslational protein modification as a result of the interaction of heme lyase with the apoprotein of cytochrome c. In mitochondria, heme lyase assists both in the transport of the polypeptide of cytochrome c through the outer membrane and in the covalent assembly of heme into the cytochrome pocket [3]. In the E. coli system the latter function can be investigated separately. Mutagenesis of cytochrome c and heme lyase is expected to provide a molecular insight into the specific interaction between these two proteins. Acknowledgement This work was supported by the Hungarian National Research Fund (OTKA T049207). References [1] Tenger, K, Khoroshyy, P., Kovács, KL, Zimányi, L, Rákhely, G (2007) Act Biol Hung in press. [2] Tenger, K, Khoroshyy, P, Leitgeb, B, Rákhely, G, Borovok, N, Kotlyar, A, Dolgikh, DA, Zimányi, L (2005) J Chem Inf Model 45: 1520-1526. [3] Dumont, ME, Ernst, JF, Sherman, F (1988) J Biol Chem 263: 15928-15937.

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23. Effects of Plant Sterols on Structure and Fluctuations of Lipid Membranes ADEN HODZIC, MICHAEL RAPPOLT, HEINZ AMENITSCH, PETER LAGGNER AND GEORG PABST. Institute of Biophysics and Nanosystems Research (IBN), Austrian Academy of Sciences, Schmiedlstr. 6, A-8042 Graz, Austria, [email protected]

We have studied the concentration dependent influence of cholesterol, stigmasterol and sitosterol on the global structure and the bending fluctuations of fluid dimyristoyl phosphatidylcholine (DMPC) and palmitoyl oleoyl phosphatidylcholine (POPC) bilayers applying small-angle X-ray scattering, as well as dilatometry and velocimetry. Applying a previously developed global X-ray data analysis technique we find that the general effect of the plant sterols is different to that of cholesterol in terms of bilayer rigidification, lateral area per lipid, increase of the membrane thickness and decrease of water layer thickness. Additional ethyl groups and double bonds of stigma- and sitosterol cause less solubility and packing in the bilayer, which is evident for higher sterol concentrations and mono-saturated bilayer (POPC). We find for both lipids that cholesterol is most efficient in increasing bilayer thickness and decreasing the bilayer rigitification, followed by sitosterol and stigmasterol. Hence, it appears that some flexibility of the sterol hydrocarbon chain is needed in order to accommodate well within the lipid bilayer. In addition, we do not observe two populations of membranes within the nominal liquid-ordered/liquid-disordered phase coexistence regime of binary sterol/lipid mixtures. This lends further support to the idea of compositional fluctuations recently brought up by fluorescence microscopy experiments which contrasts the formation of stable domains within the miscibility gap. Acknowledgement This work was supported by the Austrian Science Fund (Grant No. P17112-B10).

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24. Observation of nanometer-sized stable lipid platforms diffusing in the plasma membrane of living cells RUPRECHT, V. 1, BRAMESHUBER, M.1, MOERTELMAIER, M.1, WEGHUBER, J.1, STOCKINGER, H.2, SCHÜTZ, G.J.1 1 2

Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr.69, A-4040 Linz, Austria, [email protected] Department of Molecular Immunology, Center of Biomolecular Medicine and Pharmacology, Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria

The current picture of cellular plasma membrane is based on the existence of small stable structures which enable controlled aggregation and segregation of distinct sets of proteins. These structures, commonly termed lipid rafts, are too small and too close to be observed directly with fluorescence microscopy, too mobile for high resolution scanning techniques, and too fragile for reliable chemical purification. We developed a novel method1 (TOCCSL - Thinning Out Clusters while Conserving the Stoichiometry of Labeling) for the stoichiometric analysis of molecular aggregates in the cellular plasma membrane. We use selective photobleaching to erase all active fluorophores within a small region of the membrane, while conserving the stoichiometry of labeling in the remaining part of the membrane. At the onset of repopulation due to Brownian motion, single diffraction limited spots of individual aggregates can be resolved and quantified. To address the question of stable lipid rafts within the cellular plasma membrane, we applied TOCCSL to investigate the aggregation of a glycosyl-phosphatidyl-inositol (GPI) anchored monomeric green fluorescent protein stable expressed in living CHO cells. Besides monomers, we found a significant fraction of dimers diffusing freely in the plasma membranes. Those dimers were stable on a seconds time scale. With this study, one basis of the raft concept – the formation of stable platforms in the plasma membrane – has been confirmed.

Figure 1. Principle of TOCCSL. Anti-DNP antibodies labeled with multiple FITC molecules were used to mimic stable clusters. A fluid supported lipid bilayer containing a fraction of DNP-labeled lipid provided the matrix for the experiment. On the left, the initial equilibrium situation is shown: a surface density of ~15 clusters per µm2 makes direct observation of individual clusters impossible. Upon photobleaching for tbl=200ms, clusters were allowed to diffuse into the bleached area. To the right, three images recorded after distinct recovery times are shown: after trec=0.5ms, no fluorescence signal can be observed within the illuminated part of the membrane; this image serves as control for complete photobleaching. After trec=500ms, individual clusters were clearly resolvable in the central part of the image, indicated by the dashed white circle; such single cluster signals are used for subsequent stoichiometric analysis. Using a much longer recovery time of trec=10s, the system has nearly reached equilibrium again 1. Acknowledgement This work was supported by the Austrian Science Fund (FWF), the Competence Center for Biomolecular ResearchVienna and by the GEN-AU project of the Austrian Federal Ministry for Education, Science and Culture. References [1] Moertelmaier, M., Brameshuber, M., Linimeier, M., Schütz, G. J. & Stockinger, H. Appl Phys Lett 87, 263903 (2005).

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25. Calcium signalling in Purkinje cell somata from fish cerebellum LUKYANETZ 1, E.A., LUKYANETS 2, I.A. 1 2

Bogomoletz Institute of Physiology, NANU, Bogomoletz str,4, Kyiv, 01024, Ukraine, [email protected] International Center for Molecular Physiology, NANU, Bogomoletz str,4, Kyiv, 01024, Ukraine

Changes in intracellular calcium (Cai) play important role in signalling of cerebellar Purkinje cells, since many processes in this neurons which are involved in synaptic plasticity triggered by Ca2+ ions [1]. Activation of voltage-operated Ca2+ channels leads to increase of somatic Cai, and this signal can be amplified by calcium-induced calcium release (CICR). It was shown on mammalian Purkinje cells that they have powerful intracellular systems to control Cai,. By immunocytochemical techniques the expression of sarco-endoplasmic reticulum Ca2+ pumps (SERCA) [2], the plasma membrane Ca2+ pump (PMCA) [3] were shown in Purkinje cells. Besides, in these cells other intracellular calcium stores – mitochondria are well developed. On the other side, it is well known, that hypoxia induces the increase of intracellular calcium which following cell death in mammals. In our previous experiments on mammal neurons we found that both mechanisms are important for the development of hypoxic effect – calcium influx via calcium plasmalemmal channels and its accumulation and release from mitochondria. The initial source of such elevation is the massive influx of ions through activation of calciumof different cellular components permeable plasmalemmal channels. An important Fig. 1. Contribution in Са2+-clearance in cerebellar neurons of fish. further step is a substantial increase in calcium accumulation by mitochondria. In some species of fish the nerve system is adapted to hypoxic influences and calcium clearance can play a role in adaptation to hypoxia. The participation of all these mechanisms in calcium clearance to changes in external medium can be demonstrated in particular animal species. Therefore in our investigations we estimated the role of these organelles and other Ca2+ managers by using microfluorescent technique on cerebellar neurons of hypoxiatolerant fish Carasius auratus gibelio. We used confocal microscopy and microfluorescent technique to measure the changes in intracellular calcium concentration in isolated neurons.. In present work we studied the mechanisms responsible for Cai clearance at cerebellar cell somata and describe their respective contributions to the removal process. We found that cerebellar neurons of carasius have well expressed basic systems for cleaning of cytoplasm from Ca2+ - PMCA, SERCA, Na/Ca2+ exchanger and mitochondria, Fig.1. Indicated systems provide cleaning of cytoplasm from Ca2+ approximately in the even ratio, where SERCA and mitochondria play more prominent role. It is assumed, that Na+/Ca2+ exchanger and PMCA act greater part in support of basal level of Ca2+ in a cells, while endiplasmic reticulum and mitohondria are more involved in cleaning of cytoplasm from Ca2+ during stimulation of cell and cell signalling. Acknowledgement We acknowledge NASU for support of these studies financial support. References [1] Marty A, Llano I (1995). Curr Opin Neurobiol 5, 335-341. [2] Takei K, Stukenbrok H, Metcalf A, Mignery GA, Sudhof TC, Volpe P, & De CP (1992). J Neurosci 12, 489-505. [3] de TN, Smith CA, Wasserman RH, Beltramino C, et al. (1993). Proc Natl Acad Sci U S A 90, 11949-11953.

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26. Cone contribution to the electroretinogram in fishes inhabiting different photic environments MILOSEVIC 1, M., BAJIĆ 1, A., VISNJIC-JEFTIC 2, Z., DAMJANOVIC 3, I., ANDJUS 1, P. AND GACIC 2, Z. Faculty of Biology University of Belgrade, Studentski trg 3, Belgrade, 11001, Serbia, [email protected] Center for Multidisciplinary Studies University of Belgrade, Kneza Viseslava 1, Belgrade, 11000, Serbia 3 Institute for Problems of Information Transmission Russian Academy of Science, Ermolova str.19, Moscow, 101447, Russia 1 2

In order to show retinal photoreceptor content in fish inhabiting different photic environments we performed comparative electroretinographic (ERG) studies using flickering light stimuli that could separate rod-mediated vision (flicker fusion frequency usually less than 15Hz) from cone-mediated vision [1,2,3]. Three marine fish species (small-spotted dogfish shark, Scyliorhinus canicula, eel, Anguilla anguilla, and painted comber, Serranus scriba) and one fresh water species (goldfish, Carassius gibelio) were investigated. Our result shows (Fig. 1) that in dogfish shark b-wave amplitude significantly decreased at low frequency of stimulation and flicker fusion (frequency when the eye loses its ability to resolve individual pulses of light) was reached at 3.2 Hz. Similar effect on the b-wave amplitude was observed in the eel, but flicker fusion was reached at around 20 Hz. On the other hand, in painted comber and goldfish under intermittent light stimulation, b-waves did not change their amplitudes at low frequencies. Flicker fusion occurred at a frequency of around 25 Hz in the case of painted comber and around 33 Hz in goldfish. Observed results indicate that the dogfish shark retina contains only rods, that of the eel is rod-dominated, while goldfish and painted comber have cone-rich retinae. Acknowledgement Supported by grant #143045 and grant #143054 of the Serbian Ministry of Science and Environmental Protection References [1] Conner, JD, MacLeod, DIA (1976) Science 195: 698-699. [2] Dodt, E (1951) Nature 168: 738. [3] Frank, TM (1999) Biol. Bull. 196: 137-144.

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Fig. 1. Gain plot. Response amplitude normalized to its maximum which yields the gain, plotted vs. the stimulus frequency on a double logarithmic scale.

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27. Fusion Pores in Spontaneous Exocytosis of Peptidergic Vesicles in Lactotrophs VARDJAN 1,2, N., STENOVEC 2,1, M, JORGAČEVSKI 2, M, KREFT 1,2, M, AND ZOREC 1,2, R. 1 2

Celica Biomedical Center, 1000 Ljubljana, Slovenia Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia, [email protected]

In kiss-and-run exocytosis, the vesicle membrane reversibly fuse with the plasma membrane, It is considered that kiss-and-run exocytosis leads to full fusion upon stimulation of vesicles containing classical transmitters. However, whether this is also the case in the fusion of peptidergic vesicles is unknown. Previously we have observed that spontaneous neuropeptide discharge from a single vesicle is slower than stimulated release, owing to the kinetic constraints of fusion pore opening [1]. To explore whether slow spontaneous release also reflects a relatively narrow fusion pore, we analyzed the permeation of FM 4-64 dye and HEPES molecules through spontaneously forming fusion pores in lactotroph vesicles expressing synaptopHluorin (spH), a pH-dependent fluorescent fusion marker. Confocal imaging showed that half of the spontaneous exocytotic events exhibited fusion pore openings associated with a change in spH fluorescence, but were impermeable to FM 464 and HEPES (Fig. 1). Together with membrane capacitance measurements these findings indicate an open fusion pore diameter 70% of exocytotic events exhibited a larger, FM 464–permeable pore (>1 nm). Interestingly, capacitance measurements showed that the majority of exocytotic events in spontaneous and stimulated conditions were transient. Stimulation increased the frequency of transient events and the fusion pore dwell-time, but decreased the fraction of events with lowest measurable fusion pore [2]. Kiss-and-run is the predominant mode of peptidergic vesicles exocytosis in resting and in stimulated cells. Stimulation prolongs the effective opening of the fusion pore and expands its primary subnanometer diameter to enable hormone secretion without full fusion. Acknowledgement We thank Dr. Gero Miesenböck (Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York) for the generous gift of spH plasmid construct, and Dr. Saša Jenko and Mr. Miha Pavšič (Jožef Stefan Institute, Department of Biochemistry and Molecular Biology, Ljubljana, Slovenia) for the help in the analysis of molecular surface models.

Fig. 1. Spontaneous exocytotic events. Single vesicle signals of spH and FM 4-64 fluorescence. Fluorescence signals are plotted as the change in fluorescence (∆F) normalized to the initial fluorescence (F0) in arbitrary units. Exocytosis resulted in a rapid increase in spH fluorescence, followed by a rapid single-exponential decline (gray line) with τ of 0.7 s.

References [1] Stenovec M, Kreft M, Poberaj I, Betz W, Zorec R (2004) FASEB J 18: 1270-1272. [2] Vardjan N, Stenovec M, Jorgacevski J, Kreft M, Zorec R. (2007) J Neurosci. 27: 4737-4746.

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28. Shape changes of giant phosphatidylcholine vesicles transferred into an aqueous suspension of oleic acid PETERLIN, P.1, ARRIGLER, V. 1, SVETINA, S., 1,2 AND WALDE, P.3 University of Ljubljana, Faculty of Medicine, Institute of Biophysics, Lipičeva 2, SI-1000 Ljubljana, Slovenia, [email protected] 2 Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia 3 Department of Materials, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland 1

At a concentration exceeding the critical aggregation concentration (cac) and at a pH value around pKa, oleic acid spontaneously forms bilayer structures. This process can be greatly accelerated by the presence of pre-formed vesicles [1]; in this case, also the distribution of vesicle sizes is narrower than for the spontaneously formed vesicles, and its peak lies in the vicinity of the peak of the pre-formed vesicles. It was also found out that new vesicles form via the growth-and-division process of the existing vesicles rather than being formed de novo [2]. The catalytic effect of the pre-formed vesicles is preserved even if they are composed of a different species of lipid; in this case mixed (e.g., oleic acid/phospholipid) vesicles are formed. The experiments were conducted with large unilamellar vesicles (LUVs), and the observation relied on the dynamic light scattering which allows for a real-time monitoring of vesicle/micelle sizes, and electron microscopy which allows for a direct visual observation. The size of LUVs, however, does not allow a direct real-time visual observation. In this contribution, optical microscopy and micropipette transfer were employed to monitor the interaction of two types of vesicle systems: micrometer-sized giant unilamellar vesicles (GUVs) formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on one hand, and 100-nanometer LUVs formed from oleic acid/oleate using the extrusion technique on the other hand. Shape transformations were monitored upon a micropipette transfer of individual POPC GUVs into a chamber filled with a suspension of oleic acid/oleate LUVs. Shape transformations were found to be dependent on the concentration of oleic acid in the suspension. Below cac, initially spherical POPC vesicles started to grow long cylindrical evaginations within tens of seconds, which then started to thicken and shorten on the timescale of minutes. Above cac, a variety of effects was observed, including several consecutive transitions between evaginated and invaginated shapes. The observed effects were interpreted in the framework of the bilayer couple model [3]. Vesicle shape is a very sensitive indicator of a change in the area difference between the outer and the inner membrane leaflets, which in turn depend on the intercalation of oleic acid/oleate molecules into the outer leaflet and their translocation between the outer and the inner leaflets. Acknowledgement This work has been supported by the Slovenian Research Agency grant P1-0055 and the COST Action D27. References [1] Walde, P, Wick, R, Fresta, M, Mangone, A, Luisi, PL (1994) J. Am. Chem. Soc. 116:11649-11654. [2] Berclaz, N, Müller, M, Walde, P, Luisi, PL (2001) J. Phys. Chem. B 105:1056-1064. [3] Svetina, S, Žekš, B (1989) Eur. Biophys. J. 17:101-111.

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29. Effect of antimicrobial peptide-amide, indolicidin on biological membranes VÉGH 1, A.G., NAGY 2, K., BÁLINT 1, Z., VÁRÓ 1, GY. AND SZEGLETES 1, ZS. Inst. of Biophysics, Biological Research Center of Hungarian Academy of Sciences, Temesvari krt. 62., Szeged, H-6726 Hungary, [email protected] 2 Dept. of Medical Physics and Biophysics, University of Szeged, Dom ter 13., Szeged, H-6720 Hungary 1

Indolicidin, a cationic antimicrobial tridecapeptide amide, was isolated from cytoplasmic granules of bovine neutrophils. Its biological activity is widely studied, but detailed mechanism of action is not yet understood. We report here an in situ atomic force microscopy study of indolicidin with supported planar bilayer membranes of dipalmitoyl phosphatidylcoline (DPPC) and purple membrane of Halobacterium salinarum. Concentration dependent interaction of peptide and membranes was found. These data suggest a powerful approach in indolicidin-membrane interaction.

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30. Preparation of giant phosphatidylcholine vesicles at high ionic strength using electrical and mechanical agitation PETERLIN, P., ARRIGLER, V., AND MAJHENC, J. University of Ljubljana, Faculty of Medicine, Institute of Biophysics, Lipičeva 2, SI-1000 Ljubljana, Slovenia, [email protected]

The electroformation method [1,2], where a phospholipid film is swelling in the presence of a lowfrequency (1-10 Hz) electric field, is widely deployed for producing giant unilamellar phospholipid vesicles with diameters exceeding 10 µm, which can constitute useful models systems for biological cells. While the method is recognized as simple and reproducible technique, which appears to be efficient for various lipid mixtures, its key disadvantage is that it cannot be applied if the salt concentration in the solution exceeds 10 mM. In this contribution, we present two techniques which can help extending the usefulness of the electroformation methods towards higher ionic strengths. In the first part, we examined a recently published method [3], where the authors replace the non-ionic solution (e.g. glycerol) in the electroformation chamber with an ionic solution (e.g. NaCl) after the electroformation is completed and while the vesicles are still attached to the substrate. We noticed that the exchange of the vesicle interior, described by the authors [3], is only noticed when one of the solutions contains glycerol, and should probably be attributed to the relatively high permeability of the phospholipid membrane for glycerol. Employing other non-ionic solutions (sucrose, glucose), this phenomenon has not been observed, i.e. the exchange of the solution in the vesicle exterior produces vesicles with a transmembrane gradient. This can be employed for various purposes per se. In the second part, we tested whether in the electroformation method the electric field directly affects the formation of vesicles, or whether its influence is indirect, i.e. through the Maxwell stress tensor, which induces mechanical forces on the boundary of media with different electric properties, and which induces mechanical agitation which can be observed at the electroformation experiment. In the experiment we removed the electrodes and the electric field from the electroformation chamber and instead directly periodically mechanically agitated the substrate with the lipid film. We tested this method against the electroformation method at low ionic strength and against gentle swelling method at high ionic strength, where electroformation cannot be applied. We found out that at low ionic strength this method produces results inferior to those obtained by the electroformation method, while at high ionic strength it performs better than the same setup with no mechanical agitation. It can therefore be concluded that the electric field affects the vesicle formation both directly and indirectly. Acknowledgement Cristina del Barrios Gascón and Alain Rizkallah are acknowledged for their contribution to the experiment with mechanical agitation during their stay in our lab as part of the IFMSA student exchange during July 2006. This work has been supported by the Slovenian Research Agency grant P1-0055. References [1] Angelova, MI, Dimitrov, DS (1986) Faraday Discuss. Chem. Soc. 81:303-311. [2] Angelova, MI, Soléau, S, Méléard, P, Faucon, JF, Bothorel, P (1992) Prog. Colloid Polym. Sci. 89: 127-131. [3] Estes, DJ, Mayer, M (2005) Biochim. Biophys. Acta 1712:152-160.

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31. Biochemical and Biophysical Characterization of a Wheat Mitochondria Potassium Channel VANESSA CHECCHETTO 1, UMBERTO DE MARCHI 2, MANUELA ZANETTI 1, MARIO SOCCIO 3, MARIO ZORATTI 2, DONATO PASTORE 3, GIORGIO MARIO GIACOMETTI 1 AND ILDIKÒ SZABÒ 1 Department of Biology, University of Padova, viale G. Colombo 3. 35121 Padova, Italy, [email protected] Department of Biomedical Sciences, University of Padova, viale G. Colombo 3. 35121 Padova, Italy 3 Department of Enviromental Sciences, University of Foggia, Via Napoli, 25 - 71100 Foggia, Italy 1 2

The study of plant mitochondrial channels is still at a preliminary stage. Activities compatible with the presence of a potassium channel in durum wheat (Triticum durum Desf.) mitochondria have only recently been identified by using classical bioenergetics [1]. However, a biochemical and electrophysiological characterization of plant mitochondria as well as the molecular identification of the potassium channel is still missing. By using classical biochemical techniques we carried out an evaluation of the purity of isolated durum wheat mitochondria. To such purpose specific antibodies against mitochondrial cytochrome c and against specific proteins of the plasma membrane and of other organelles have been used, demonstrating that the mitochondria isolated by us were slightly contaminated only by endoplasmatic reticulum. In order to gain information about the molecular identity of the potassium channel, we used a specific antibody against the highly conserved pore region of all potassium channels and identified distinct bands that were specifically recognized. Mass spectrometry analysis of these bands is under way. The functional characterization of the channel activities in wheat mitochondria was performed by using the patch clamp electrophysiological technique, never used before on plant mitochondria. Preliminary experiments reveal the presence of a potassium-selective channel with a conductance of 100 pS in the inner mitochondrial membrane. References: [1] Pastore et al (1999), J. Biol. Chem. 274, 26683-26690.

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32. Quantitative Characterization of the Large Scale Association of ErbB1 and ErbB2 by Flow Cytometric Homo-FRET Measurements ÁGNES SZABÓ 1, JÁNOS SZÖLLİSI 1,2, AND PETER NAGY 1 1 2

Department of Biophysics and Cell Biology, University of Debrecen, 1 Egyetem sqr, Debrecen, 4010, Hungary, [email protected] Cell Biophysical Work Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, University of Debrecen, 1 Egyetem sqr, Debrecen, 4010, Hungary

The epidermal growth factor (EGF) receptor family of receptor tyrosine kinases comprises four members: ErbB1 (EGFR), ErbB2, ErbB3 and ErbB41. They are present in low quantities on the surface of epithelial cells, but they are often overexpressed in cancer cells. Their activation involving the ligand-induced formation of receptor homo- and heterodimers stimulates a cascade of intracellular kinases leading to migration, cell survival and proliferation. ErbB2 is a ligand-less member of the ErbB family that functions as the preferred coreceptor for the other ErbB proteins. At high expression level it can be activated by ligandindependent homoassociation. ErbB3 has no intrinsic kinase activity transmitting activating signals only in heterodimers. Thus, the association of ErbB receptors is the first step in the activation cascade. Although previous measurements revealed that extensive associations are present (approximately a thousand ErbB2 proteins in a homocluster)2, only dimers have been investigated in detail, because they are amenable to biochemical approaches. For further characterization of large-scale clusters of ErbB1 and ErbB2 we did flow cytometric homo-FRET (FRET = fluorescence resonance energy transfer) measurements. In homo-FRET the interaction takes place between two spectroscopically identical fluorophores (labeled antibodies). A homocluster contains a collection of identical, fluorophore-labeled molecules, each of which is able to interact with the others directly or indirectly by homo-FRET. Therefore, the excitation energy is distributed to the ensemble of molecules by homo-FRET. The only manifestation of homo-FRET is decreased fluorescence anisotropy 3. We have developed a method for studying the dependence of fluorescence anisotropy on the concentration of labeled antibodies. We assumed that a fraction of proteins is monomeric, and the rest is present in clusters of N-mers. The model was used to fit the anisotropy data yielding the fraction of monomers and the size of the clusters. First, we determined the cluster size and monomer% of ErbB1 and ErbB2 in quiescent cells, and found that the cluster size of ErbB2 was significantly bigger than that of ErbB1. Stimulation of cells with EGF (ligand of ErbB1) and heregulin (ligand of ErbB3) lead to a decrease in the cluster size of ErbB2. We attribute this phenomenon to the recruitment of ErbB2 to heterodimers with ErbB1 and ErbB3 resulting in the removal of ErbB2 from homoclusters. On the contrary, stimulation of ErbB1 with EGF lead to an increase in the cluster size of ErbB1 in cells with high ErbB1 and low ErbB2 levels, because ErbB1 was activated by ligand-induced homodimerization. These findings point to the fundamentally different ways of activation followed by ErbB1 and ErbB2. We determined the level of activation of ErbB2 in quiescent and stimulated cells, and found a negative correlation between the cluster size and the activation state of the receptor implying that ErbB2 homoclusters contain inactive ErbB2 molecules. We have successfully used our method for the quantitative characterization of a new dimension of receptor associations which can only be investigated by homo-FRET measurements. Acknowledgement Grant support: OTKA F049025. References [1] Yarden, Y. et al. (2001) Nature Reviews Molecular Cell Biology 2: 127-37. [2] Nagy, P et al. (1999) Journal of Cell Science 112: 1733-41. [3] Runnels, LW et al. (1995) Biophysical Journal 69: 1569-83.

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33. The Use of EPR to Determine the Plasma Membrane Dynamics of Xanthophylomyces Dendrorhous and the Effect on Its Various Carotenoid Types BLASKÓ 1, A., BELÁGYI 2, J., LİRINCZY 2, D. AND PESTI 1, M. 1 Department

of General and Environmental Microbiology, Faculty of Sciences, University of Pécs, P. O. Box 266, H-7601 Pécs, Hungary, [email protected] 2 Institute of Bioanalysis, Medical Faculty of Pécs University, Hungary

The carotenoids present in Xanthophylomyces dendrorhous prevent external oxidative damage to the plasma membrane. They have differing properties (polar, non-polar) which variously build lipid chains into the plasma membrane, forming differing binding forces. As a result the membrane`s structure dynamic is altered. We used the spin-labelling method to study the various properties of the plasma membrane dynamics of Xanthophylomyces dendrorhous in relation to temperature. In the case of fluid-phase membranes, it was found that polar carotenoids, such as astaxanthin and cis-astaxanthin, increase the electro paramagnetic resonance (EPR) order parameter and decrease the motional freedom, phase-transition temperature and the flexibility gradient of the alkyl chains of the lipids, as shown with stearic acid spin labels. In contrast, the non-polar carotenoids β-cryptoxanthin and β-carotene decreased the EPR order parameter and increased motional freedom, phase-transition temperature and the flexibility gradient of alkyl chains.

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34. Phase seperation and the shape of giant phospholipid vesicles MAJHENC, J., ARRIGLER, V., SVETINA S., AND ŽEKŠ B. University of Ljubljana, Faculty of Medicine, Institute of Biophysics, Lipičeva 2, J. Stefan Institute, Jamova 9, SI-1000 Ljubljana, Slovenia, [email protected]

Ternary mixtures of high melting temperature lipid, low melting temperature lipid and cholesterol form liquid order and liquid crystal domains [1,2]. Giant vesicles were prepared from DPPC/DOPC/cholesterol mixture in 0.2 M sucrose by electro-formation method at the temperature higher than 350C. All further experiments were preformed at room temperature. Domains on giant vesicles were directly observed by fluorescence microscopy. NBD (green) marked lipid and BODIPY (red) marked cholesterol where used to identify the domains. Interaction with Triton-100 and consequent shape changes and tether formation from liquid order domain of a vesicle membrane was used to determine the dominant marker in liquid order (NBD) and liquid crystalline phase (BODIPY). Incorporation of Triton-100, mostly in liquid crystalline domain induces the decrease the border line between the domains. The division of a vesicle was also observed with completely separated new formed vesicles. Vesicle with membrane which was composed from only two domains, one in liquid order and one in liquid crystal phase was chosen because of simple geometry and more convenient comparison with theory. Using micro-injector, the volume of vesicle was gradually decreased or/and increased by changing the osmolarity of sugar solution. The shape of a vesicle for each change of a volume was followed on the phase contrast and the two fluorescence image. The obtained shape sequences are compared with model predictions, where either line tension between the domains or different value for Gauss curvature module are assumed. References [1] Bagatolli, LA, Gratton, E. Biophys. J. 78:290-305. [2] Veatch, SL, Keller, SL (2003) Biophys. J. 85:3074-3083. [3] Baumgart, TB, Hammond, AT, at all (2007) PANS 104:3165-3170.

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35. Reversal of multidrug resistance by rerouting of doxorubicin from the cytoplasm into the nucleus mediated by a new family of polyglycerol-based copolymers TATIANA V. DEMINA 1,5, JÖRG NIEBERLE 2, PETER POHL 3, HOLGER FREY 2, ALEXANDER A. SHTIL 4, IRINA D. GROZDOVA 1, NICKOLAY S. MELIK-NUBAROV 5,*, ELENA E. POHL 1,* ,@ 1Institute

of Cell Biology and Neurobiology, Charité-Universitätsmedizin, Berlin, Germany. @[email protected] Institute of Organic Chemistry, Johannes Gutenberg-University, Mainz, Germany 3 Institute of Biophysics, Johannes Kepler University, Linz, Austria 4 Blokhin Cancer Research Center, Moscow, Russia 5 Moscow State University, Chemistry Department, Moscow, Russia, [email protected] 2

Multidrug resistance (MDR) of tumor cells is a serious obstacle in cancer therapy. Chemosensitization by novel PPO-PG amphiphilic block copolymers, a class of compounds structurally close to MDR reversing Pluronics®, is a promising approach because of the high efficiency and relatively low toxicity of these polymers. However, the underlying mechanism is under dispute. To distinguish between (i) direct Pgp polymer interactions and (ii) unspecific effects like disturbances of the membrane barrier, we investigated a series of homologues copolymers, based on hyperbranched polyglycerol, which differed only in the size of the hydrophilic blocks. The observed resensitization of resistant cells (MCF7/DOX, K562/DOX and K562/iS9) cannot be attributed to an increase in doxorubicin influx since the plasma membrane permeability remained < 1 nm/s. Rather it has to be attributed to the copolymer-mediated redistribution of doxorubicin from intracellular stores into the nucleus as revealed by two-photon laser scanning microscopy. The similarities in the effects of hydrophobic and hydrophilic copolymers suggested that the homologues shared a common intracellular pathway. Most likely, specific binding to a protein was responsible. Together these results imply that the block copolymers act to specifically inhibit Pgp in the membranes of intracellular organelles. Acknowledgement The authors express their gratitude to the Volkswagen Stiftung, the Russian Foundation for Basic Research (Grant 0603-32403 and SS-602.2003.3), Charite-Universitätsklinikum, Berlin, Germany (2003-083), German Academic Exchange Service (DAAD A/04/000154 to TD) for financial support of this work. H. F. is grateful to the Fonds der Chemischen Industrie (FCI).

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36. CRAC channels in developing human dendritic cells PAPP 1, F., HAJDU 1, P., VARGA 1, Z., ZSIROS 1, E., LUDÁNYI 2, K., GÁSPÁR 1,3, R., RAJNAVÖLGYI 2,3, É., AND PANYI 1,3, G. University of Debrecen, Dept. of Biophysics and Cell Biology, 98. Nagyerdei krt., Debrecen, 4012, Hungary, [email protected] University of Debrecen, Dept. of Immunology, 98. Nagyerdei krt., Debrecen, 4012, Hungary, 3 Research Center for Molecular Medicine University of Debrecen, 98. Nagyerdei krt., Debrecen, 4012, Hungary, 1 2

According to their phenotype, function and tissue localization two types of dendritic cells (DCs) can be distinguished. Immature DCs (IDCs) make the first contact with foreign antigens in the periphery (e.g. skin) where they uptake many different kinds of substance using non-specific (pinocytosis, macropynocytosis) and specific (receptor-mediated phagocytosis) pathways and process them. When an infection occurs, they are stimulated to migrate via the lymphatics to the local lymphoid tissues, where they have a completely different, “mature” phenotype (MDCs); the expression of MHC and other co-stimulatory molecules enables them to stably present peptides from proteins acquired from the infecting pathogens. The signals that activate tissue DCs to migrate and mature after taking up antigen are clearly of key importance in determining whether an adaptive immune response will be initiated. Among other signaling pathways the role of the cytosolic free Ca2+ ([Ca2+]i) in the differentiation and function of DCs is recognized. Chemokines, for example, induce a rise in the [Ca2+]i suggesting that [Ca2+]i might regulate the migration of DCs. Ca2+ chelators applied extracellularly inhibit the maturation of DCs, including the expression of MHC-II and that of the co-stimulatory molecules. On the contrary, [Ca2+]i ionophores alone are sufficient to induce the maturation of DCs in the absence of appropriate cytokines. Furthermore, the differentiation of DCs into DC1 and DC2 phenotypes, being responsible for the activation of Th1 and Th2 T cells, respectively, can be triggered by the selective induction of the Ca2+ signal. To date, however, little is known about the mechanisms being responsible for the rise in the [Ca2+]i in DCs in general, an in human DCs in particular. Human dendritic cells at different stages of differentiation were produced in vitro from freshly isolated human monocytes using a combination of cytokines according to the procedure developed in our laboratories. The [Ca2+]i was measured using FURA-2 in ratiometric mode of a PTI Delta Scan microscope fluorimeter. Calibration of the fluorescence signal to [Ca2+]i was performed using a commercially available calibration kit (SIGMA). Ionic currents through CRAC channels were measured in the whole-cell mode of patch-clamp using voltage ramps. The pipette filling solution was based on Cs-methansulfonate supplemented with 10 mM Mg2+ to inhibit MIC currents. Blockers of CRAC channels and solutions of different ionic compositions were delivered into the recording chamber using a gravity-driven computer controlled perfusion system. Recording of ionic currents in voltage clamped IDCs and MDCs failed to identify any voltage-gated Ca2+ conductance. On the contrary, depletion of the intracellular Ca2+ stores using the SERCA ATP-ase inhibitor thapsigargin (1 µM) evoked a biphasic Ca2+ signal in both IDCs and MDCs. Ion substitution experiments showed that the second phase of the signal is absent 0 mM extracellular Ca2+ characteristic of the activation of CRAC channels. Pharmacological experiments using CRAC blockers SKF-96365 (20 µM) and2-APB (40 µM) inhibited the Ca2+ signal generated by the depletion of intracellular stores. These experiments pointed to CRAC channels as the primary channels being responsible for Ca2+ signaling in both IDCs and MDCs. These findings were confirmed using patch-clamp where a membrane conductance with high selectivity for Ca2+ over Cs+ was evoked by the depletion of the intracellular stores using BAPTA-containing pipette filling solution. In summary we showed that CRAC channels are expressed in both immature and mature dendritic cells. The characterization of their expression level and their activation by signals leading to the maturation of the cells is needed to substantiate the physiological role of CRAC channels in dendritic cell development. Acknowledgement This work was supported by the following grants: OTKA NK 61412, K 60740; ETT/076/2006, 064/2006 and 068/2006.

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37. Coexpression of IL9R with IL2R superclusters of T lymphoma cells

and

MHC

glycoproteins

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common

NIZSALÓCZKI 1, E., VÁMOSI 1, G., FAZEKAS 1, Z., CSOMÓS 1 I., SZALÓKI 1, N., GOLDMAN 2, C.K., WALDMANN 2, T. A., DAMJANOVICH 1, S. AND BODNÁR 1, A. Cell Biology and Signaling Research Group of Hung. Acad. Sci., Department of Biophysics and Cell Biology, University of Debrecen, Nagyerdei krt. 98, Debrecen, 4012, Hungary, [email protected] 2 Metabolism Branch, NCI, National Institutes of Health, 20892, Bethesda, USA 1

IL-9 is a multifunctional cytokine with pleiotropic effects on T cells. Biological effects of IL-9 are mediated by its heterodimeric receptor complexes consisting of the cytokine-specific alpha subunit and the common gamma chain shared with other cytokines including IL-2 and IL-15, important regulators of T cell function. Previously we have shown preassembly of the heterotrimeric IL-2 and IL-15 receptors as well as association of their alpha chains in human T lymphoma cells. Binding of IL-2 or IL-15 altered interactions within the receptor complexes [1,2]. We have also demonstrated that IL-2 and IL-15 receptors form supramolecular clusters with MHC glycoproteins in lipid rafts of T cells [1,3]. Here we investigated cell surface organization of IL-9R in Kit225 T lymphoma cells transfected with IL9Ralpha. By using CLSM and FRET we have shown co-expression and molecular scale association of IL9Ralpha with IL2R and MHC molecules in common membrane domains (lipid rafts) suggesting that IL9Ralpha is another component of the aforesaid superclusters. Effect of cytokine treatment on the interactions of IL-9Ralpha with the shared gamma chains (assembly of IL-9R) as well as with other subunits of IL-2R was also studied. According to our data arrangement of the IL-9/IL-2R subunits can be modelled with a heterotetrameric structure which is modulated upon cytokine binding. This arrangement would allow efficient sharing of the gamma chains by the two receptor kinds. Acknowledgement

This work was supported by the following grants: OTKA NK61412, F46497, T48745; ETT 065/2006, 070/2006; NATO Life Science and Technology Collaborative Linkage Grant 980200 and Bolyai Janos Research Fellowships (to A. Bodnar and G. Vámosi). References [1] Vámosi G, Bodnár A, Vereb G, Jenei A, Goldman CK, Langowski J, Tóth K, Mátyus L, Szöllösi J, Waldmann TA, Damjanovich S. (2004) Proc Natl Acad Sci USA 101: 11082-11087. [2] Damjanovich S, Bene L, Matkó J, Alileche A, Goldman CK, Sharrow S, Waldmann TA. (1997) Proc Natl Acad Sci USA 94: 13134-13139. [3] Matkó J, Bodnár A, Vereb G, Bene L, Vamosi G, Szentesi G, Szollosi J, Gaspar R, Horejsi V, Waldmann TA, Damjanovich S. (2002) Eur J Biochem 269: 1199-1208.

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38. A prokaryotic potassium channel is involved in the regulation of photosynthesis in cyanobacteria. MANULEA ZANETTI, ELISABETTA BERGANTINO, NICOLETTA LA ROCCA, ENRICO TEARDO, GIORGIO MARIO GIACOMETTI AND ILDIKÓ SZABÓ University of Padua, Department of Biology, viale G. Colombo 3, Padova, 35121-Italy, [email protected]

A putative potassium channel (SynKCh) has been identified in the genome of the cyanobacteria Synechocystis sp. PCC6803, a photoheterotroph organism, considered to be the precursor of chloroplasts. SynKCh was cloned from Synechocystis and expressed in eukaryotic cells. Electrophysiological analysis demonstrated that in this heterologous expression system the protein gives rise to outwardly rectifying, potassium selective current. In order to investigate the physiological role of this channel, a SynKCh-deficient Synechocystis mutant was obtained which was unable to grow in normal conditions; however growth could be restored in the presence of an artificial electron donor, under high-intensity light illumination or in the presence of added carbon source. Western blot, performed using an antibody raised against recombinant SynKCh, revealed that expression of the channel protein was regulated by the redox-state. Immunogold electron microscopy showed localization of SynKCh in thylakoid membranes in cyanobacteria. Pulse amplitude modulated fluorimeter experiments indicated that SynKCh-deficient cyanobacteria are unable to build up proton gradient across thylakoid membrane upon illumination. These data suggest that the channel may be crucial for regulation of photosynthesis.

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39. Organization of the interleukin-2 and -15 receptor subunits (IL2α-IL15α) on resting Kit225 FT7.10 T cells DE BAKKER 1, B.I,

BODNÁR 2, A., VAN DIJK 1, E. M.H.P., VARGA 3, S., VÁMOSI 2, G.,DAMJANOVICH 2, S., MÁTYUS 4, L., VAN HULST N.F., JENEI 4, A., AND GARCIA-PARAJO 6, M.F. 5,

Applied Optics group, Faculty of Science and Technology, MESA+ Research Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands; 2 Cell Biophysics Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, 4012 Debrecen, Hungary. 3 Clinical Research Center, Medical and Health Science Center, University of Debrecen, 4012 Debrecen, Hungary. 4 Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, P.O. Box 39, 4012 Debrecen, Hungary. [email protected] 5 ICFO-Institut de Ciències Fotòniques, 08860 Barcelona, Spain and ICREA-Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain 6 IBEC-Institut de Bioenginyeria de Catalunya & CIBER-BNN, Barcelona Science Park (PCB), Josep Samitier 1-5, Barcelona 08028, Spain and ICREA-Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain.. 1

Interleukin-2 and -15 (IL-2 and IL-15) could provide quite distinct contributions to T cell mediated immunity despite their very similar receptor composition and signaling machinery. Since most of the proposed mechanisms underlying this apparent paradox attribute key significance to the private α-chains of IL-2 and IL-15 receptors, we investigated spatial organization of IL-2Rα and IL-15Rα on a human CD4+ leukemia T cell line, Kit225 FT7.10 by using Scanning Near-Field Optical Microscopy (SNOM) and Transmission Electron Microscopy (TEM). In addition to results obtained with other methods reporting on the lateral distribution and co-localization of IL-2Rα and IL-15Rα on the few-hundred-nanometer scale (e.g. confocal laser scanning microscopy), single molecule sensitivity of our SNOM instrument utilized in this study can also provide insights to the inner structure of these domains. Our SNOM experiments revealed that both types of α-subunits exist on the cell surface either in large (several-hundred-nanometer size) domains or as individual entities/small clusters. Whereas only a minor fraction of IL-2Rα resided outside the domains, as many as ~30% of IL-15Rα chains were found as individuals or small clusters, excluded from the main domain regions. The IL-2Rα and IL-15Rα domains had an average diameter of ~450 and ~360 nm, respectively, and exhibited a constant packing density, i.e. the number of α-chains accommodated by them was linearly proportional to the size of the domains. Dual color SNOM experiments as well as TEM investigations showed co-clustering of the two types of α-chains. The analysis of our data may contribute to our understanding the action of the IL-2/IL-15 receptor system in T cell function and also to the more rationale design of IL-2R- and IL-15R-targeted immunotherapy of human leukemia.

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40. Small cationic outward rectifier from plasma membrane of fungus Phycomyces

blakesleeanus (Burgeff) ŽIVIĆ 1, M., POPOVIĆ 2, M. AND VUČINIĆ 2, Ž. 1 2

Institute of General and Physical Chemistry, Studentski trg 12-16, Belgrade, 11000, Serbia Center for Multidisciplinary Studies, Bulevar despota Stefana 142, Belgrade, 11060, Serbia

Studies of ion channels in filamentous fungi are still in early stages. Only three ionic channels have been completely characterized [1, 2, 3]. This comes as a consequence of difficulties encountered in formation of a high resistance seal –“giga-ohm seal” (>1GΩ) between the hyphal plasma membrane and patch clamp pipette, that is due to the existence of the external cell wall and problems encountered with its removal. Membrane of citoplasmatic droplets obtained from sporangiophores of fungus Phycomyces blakesleeanus, which is proposed as a system for characterization of plasma membrane ion channels from filamentous fungi [4], was examined by patch clamp techniques. Its most conspicuous feature, in the absence of energizing substrates, is a cation channel with a characteristic conductance of 10.5 ± 0.2 pS for symmetric 125 mM KCI solutions. The channel was recorded in 40% of conducted patches, with more than 10 active channels per patch. It has little selectivity between monovalent cations (PNa / PK / PCs ≈ 1) but strong selectivity for monovalent cations over divalent cations (PCa /PK = 0.025). Channel gating is voltage-dependent; open probability, P., reaches maximum (1.0) at a transmembrane voltage of 120 mV (cytoplasmic surface negative) and declines at more negative voltages (i.e., to 0 around -50 mV). The time-averaged currentvoltage curve shows strong outward rectification, with positive currents (positive charges flowing from cytoplasmic side to extracellular side) much larger than negative currents. Acknowledgement

This work was supported by a grant from the Ministry of Science and Environmental Protection of the Republic of Serbia (projects No. 143016B). References [1] Roberts, SK (2003) Eukaryot. Cell 2: 181-190. [2] Roberts, SK, Dixon, GK, Dunbar, SJ, Sanders, D (1997) New Phytol. 137: 579-585. [3] Zhou, X-L, Stumpf, MA, Hoch, HC, Kung, C (1991) Science 253: 1415-1417. [4] Živić, M, Popović, M, Živanović, B, Vučinić Ž (2005) Ann. N.Y. Acad. Sci. 1048: 491-495.

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41. A Calcium Switch Triggers Formation of Inactive Factor Xa Dimers on Phosphatidylserine Containing Membranes KOKLIČ 1,2, T. WEINREB 1, G.E. MAJUMDER 1, R. AND LENTZ 1, B.R. 1 2

Univ. of North Carolina at Chapel Hill, Dept. Biochem. & Biophysics, CB#7260, 101 Manning Dr, Chapel Hill, NC 27599-7260, USA. Jožef Stefan Institute, Jamova 39, Ljubljana, SI-1000, Slovenia, [email protected]

Factor Xa has a prominent role in amplifying both inflammation and coagulation cascades. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. PS, in the presence of Ca2+, triggers tight association of factor Xa with its cofactor, factor Va. An interesting complication is that PS also triggers tight association of factor Xa with factor Xa, at least in solution [1], to form an inactive factor Xa dimmer [2]. In this work, we ask whether Ca2+ and PS also trigger formation of an inactive factor Xa dimer on a membrane and explore the possible physiological significance of this. We have determined the proteolytic activity of human factor Xa towards human prethrombin2 as a substrate both at fixed membrane concentration (increasing factor Xa concentration) and at fixed factor Xa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as factor Xa bound to membranes. The total factor Xa activity actually decreased as low concentrations of PS-containing membranes were added, and increased at higher membrane concentrations. At fixed membrane Fig. 1. Response of factor Xa activity to addition of PS membranes at different calcium concentrations. concentrations, the total factor Xa activity did not increase proportionally with factor Xa concentration. Both observations suggested the existence of membrane-bound and inactive multiimeric forms of factor Xa. Because we have observed factor Xa to form dimers in solution [1], we tried to fit globally four such data sets to a model that takes into account dimerization of factor Xa after binding to a membrane. This dimer model successfully described all our data, with the parameters of best fit being kcat/KMdimer = 0 M-1s-1, kcat/KMmonomer = 7000 M-1s-1, kcat/KMsolution = 38 M-1s-1, and Kd,surfacedimer = 2·10-14 mol/(dm)2. Also consistent with the dimer hypothesis, we observed that the binding of factor Xa to PS-containing membranes appears to be tighter at low than at high membrane concentration. As we observed for soluble-PS-induced dimer formation in solution, dimer formation on a membrane was Ca2+ dependent. Unlike in solution, factor Xa was activated by membrane binding below 1.1 mM Ca2+, but inactivated above this Ca2+ concentration. This suggests that factor Xa activity may be regulated by Ca2+ concentrations close to plasma Ca2+ levels. We conclude that: 1) factor Xa dimerizes on PS-containing membranes; 2), factor Xa dimer is inactive; and 3), the transition from monomer to dimer state depends critically on Ca2+ concentration. Acknowledgement Supported by USPHS grant HL072827. References [1] Majumder, R, Wang, JF, Lentz, BR (2003) Biophys J 84:1238-1251. [2] Sen, S, Lentz, BR, unpublished

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42. The role of lysophospatidic acid in amyloid formation of β2-microglobulin under physiological conditions PÁL-GÁBOR 1,2, H., GOMBOS 1, L., MICSONAI 1, A., KOVÁCS 2, E., KOVÁCS 1, J., GRÁF 1, L., GOTO 3, Y., LILIOM 2, K., AND KARDOS 1, J. Institute of Biology, Eötvös Loránd University, Pázmány sétány 1/C, Budapest, H-1117, Hungary, [email protected] Institute of Enzymology, Hungarian Academy of Sciences, Karolina út 29, Budapest, H-1113, Hungary 3 Institute for Protein Research, Osaka University and CREST, Japan Science and Technology Agency, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan 1 2

Acknowledgement J.K. is a grantee of the Bolyai János Scholarship of the Hungarian Academy of Sciences. This work was supported by the Hungarian National Science Foundation (OTKA 61501 and TS049812) and the Medical Research Council (ETT 555/2006).

A ThT fluorescence (a.u.)

β2-microglobulin (β2m), a protein responsible for dialysisrelated amyloidosis [1], readily forms amyloid fibrils in vitro at pH 2.5 in a seed-dependent manner [2]. However, under physiological conditions, it cannot polymerize without additives even at elevated concentrations [3,4]. It has not yet been fully understood which factors evoke β2m amyloid formation in vivo. In this work we show that lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid, promotes amyloid formation through a complex mechanism. Fluorescence and circular dichroism spectroscopy, differential scanning calorimetry, electron microscopy, and limited proteolysis experiments showed that LPA, at a concentration as low as 50 µM, destabilizes the structure of the native monomer β2m inducing a partially unfolded intermediate state. This intermediate is in equilibrium with native β2m and capable of fibril extension upon addition of preformed fibril seeds to the solution. Moreover, LPA stabilizes the structure of β2m fibrils as revealed from thermal denaturation studies. Negative control measurements with other phospholipids such as lysophosphatidylcholine (LPC), sphingosylphosphorylcholine (SPC), and sphingosine-1-phosphate (S1P) has proved that LPA establishes specific interactions with β2m.

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Fig. 1. (A) Amyloid fibril formation in the presence of LPA and preformed seeds at pH 7.5 and 37 ºC. Fibril growth was monitored by ThT fluorescence. Samples contained 300 µM (●) or 0 µM (▲) LPA. (B) TEM image of β2m amyloid fibrils formed in the presence of LPA. The sample was stained with 1% uranyl acetate. The scale bar represents 300 nm.

References [1] Geyjo, F, Yamada, T, Odani, S, Nakagawa, Y, Arakawa, M, Kunitomo, T, Kataoka, H, Suzuki, M, Hirasawa, Y, Shirahama, T, et al. (1985) Biochem. Biophys. Res. Commun. 129: 701-706. [2] Naiki, H, Gejyo, F (1999) Methods Enzymol. 309: 305-318. [3] McParland, VJ, Kad, NM, Kalverda, AP, Brown, A, Kirwin-Jones, P, Hunter, MG, Sunde, M, Radford, SE (2000) Biochemistry 39: 8735-8746. [4] Yamamoto, S, Hasegawa, K, Yamaguchi, I, Tsutsumi, S, Kardos, J, Goto, Y, Gejyo, F, Naiki, H (2004) Biochemistry 43: 11075-11082.

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43. Molecular probe to measure the electrostatic potential inside proteins GERENCSÉR L.1, TAKAHASHI E.2, DERRIEN V.3, SEBBAN P.3, WRAIGHT C.2, HANSON D.4 AND MARÓTI P.1 1 Department

of Biophysics, University of Szeged, Szeged; [email protected] of Biochemistry and Center for Biophysics and Computational Biology, University of Illinois, Urbana; 3 Laboratoire de Chimie-Physique, Université Paris-Sud, Orsay; 4 Biosciences Division, Argonne National Laboratory, Argonne 2 Department

The reaction centre (RC) of bacterial photosynthesis is well-known energy-transducing membrane protein. Its structure was determined by X-ray diffraction analysis with the highest resolution of 2.2 Å. The light-excitation initiates intraprotein electron transfer along pigment molecules that are strongly attached to the protein scaffold. The electron transfer is coupled with proton uptake and finally the reduced and oxidised equivalents are exported at the cytoplasmic and periplasmic site of the membrane. The electrostatic potential inside the protein controls the rate of electron transfer and thus the photosynthetic activity of the bacterium. The potential field around the RC dissolved in aqueous solution can be calculated with numerical methods using structural and charge distribution data. As there are several problems and heuristic assumptions in the calculations, therefore we intend to determine the electrostatic potential by experimental methods. The non-redox active inhibitors block the interquinone electron transfer by binding with large affinity to the secondary quinone (QB) binding site of the RC. The antibiotic inhibitor, stigmatellin possesses a phenol group and its degree of protonation (pK) is highly sensitive to the variation of the electrostatic potential in the environment of the QB pocket. This pK shift can be followed with changes in the steady state absorption spectrum of the stigmatellin. In the vicinity of QB binding site several ionisable residues are located and their substitutions lead to the modulation of the potential field. In the RC of single site-directed mutant of L213Asp→Ala the pK of the stigmatellin dropped by 3.5 pH units to 7.3 from the value of the wild type RCstigmatellin complex. The replacement of second acidic residue by neutral one (L212Glu-L213Asp→AlaAla) did not given rise further decrease of the pK, which means that one of these two residues (L212Glu and L213Asp) is protonated even at neutral pH. The contributions of individual protonatable groups to the measured electrostatic potential are planned to determine with the effective method of combination of site-directed mutagenesis and binding of stigmatellin, which acts as electrostatic probe. Furthermore we propose to find a molecular probes with lower pK that are more suitable to monitor the potential decrease as well or different type of probes, which are not specific to the quinone binding site.

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44. Tuning of the electron transfer rate by the altered redox potential of heme c in cytochrome c and in its complex with cytochrome c oxidase KHOROSHYY 1, P., TENGER 1, K. AND ZIMÁNYI 1, L. 1 Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62., Szeged, H-6701, Hungary, [email protected]

The photoinduced covalent redox label 8-Thiouredopyrene-1,3,6-trisulfonate (TUPS) [1] has been attached to a cysteine residue introduced by site directed mutagenesis replacing Ala15 of horse heart cytochrome c. Electron transfer between TUPS and the heme of cytochrome c was measured spectroscopically. The forward electron transfer was found to be very fast (submicrosecond), whereas the reverse electron transfer was 10 µs at room temperature. A second site directed mutation replacing the axial heme ligand Met80 with His resulted in a large negative shift of the heme redox potential, as expected. Electron transfer measurements on the A15C/M80H double mutant indicated substantial acceleration of the reverse electron transfer (submicrosecond) and deceleration of the forward electron transfer (~ 20 µs), due to the altered driving forces for these processes. Ascorbate cannot reduce the heme of the double mutant, but serves as an electron donor to the triplet excited state of TUPS [2]. In the presence of ascorbate a different process was observed: the TUPS triplet was converted to the TUPS negative radical, which then reduced heme c much more efficiently than in the absence of ascorbate. Both the A15C and the A15C/M80H mutant, labeled cytochromes as well as labeled yeast iso-1 cytochrome c were complexed with bovine heart and with Paracoccus denitrificans cytochrome c oxidase (COX) to follow electron transfer through the putative physiological route. Various efficiencies of COX reduction were observed. An apparent electron deficiency was assigned to the spectrally silent reduction of CuA, and the reduction of heme a was detected spectrally. Acknowledgement Supported by the Hungarian Scientific Research Fund (OTKA T049207). References [1] Kotlyar, A.B.; Borovok, N.; Hazani, M. Biochemistry 1997, 36(50), 15828-15833. [2] Kotlyar, A.B.; Borovok, N.; Khoroshyy, P.; Tenger, K.; Zimányi, L. Photochem. Photobiol. 2004, 79(6), 489-493.

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45. Alternative Respiration of Fungus Phycomyces blakesleeanus (Burgeff) during development ZAKRZEWSKA 1, J., ŽIVIĆ 1, M., STANIĆ 2, M. AND ŽIVANOVIĆ 3, B. Institute of General and Physical Chemistry, Studentski trg 12-16, Belgrade, 11000, Serbia Faculty of Biology, University of Belgrade, Studentski trg 3, Belgrade, 11000, Serbia 3 Center for Multidisciplinary Studies, Bulevar despota Stefana 142, Belgrade, 11060, Serbia 1 2

Fungal respiratory chain is highly diverse and for the most of fungi insufficiently investigated. In this study the respiration of fungus Phycomyces blakesleeanus has been measured by means of Clark-type oxygen electrode. We show that P. blakesleeanus, apart of classical cytochrome respiratory pathway (cyanide-sensitive respiration, CSR), posses also alternative respiration (cyanide-resistant respiration, CRR). Proteins performing CSR and CRR are cytochrome c- and alternative oxidase (COX and AOX), respectively. The activities of COX and AOX were determined in spores and mycelia at different developmental stages (12 – 40 h-old) by monitoring oxygen uptake in the presence of respiratory inhibitors, cyanide (KCN) and/or salicylhydroxamic acid (SHAM). Alternative respiration of P. blakesleeanus is constitutively expressed through development and is an active component of the electron transport chain. The intensity of CRR decreased during development, being the highest in activated spores. The addition of KCN to activated spores increased total respiration, whereas subsequent treatment with SHAM caused complete inhibition of respiration. The lowest level of CRR, detected in 20h-old mycelia, could be associated with a change in its environment, starting from low oxygen water medium to its high oxygen level at the surface of the medium. The intensity of alternative respiration was higher when COX was previously inhibited, which implies that only a fraction of AOX, present in the inner mitochondrial membrane, is active during respiration under standard growing conditions. We show that alternative respiration was stimulated in the presence of antimycin A in the bathing medium, while subsequent addition of cycloheximide, (cytosolic translation inhibitor) or carbonylcyanide-mchlorophenylhydrazone (uncoupler) blocked this effect. Actinomycin D (transcription inhibitor) did not affect alternative respiration. Presented results argue in favour of de novo synthesis of AOX protein in cytosol of mycelium transported to mitochondria, which is controlled on the translational level. Acknowledgement

This work was supported by a grant from the Ministry of Science and Environmental Protection of the Republic of Serbia (projects No. 143016B).

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46. Statistical method to resolve fluorescence emission spectra measured from dark-grown pea seedlings SZENZENSTEIN*, A., KÓSA, A. AND BÖDDI, B. Department of Plant Anatomy, Institute of Biology, Eötvös University, Pázmány P. s. 1/C, Budapest H-1117, Hungary [email protected]

Chlorophyll-type pigments form various pigment-protein complexes in vivo, which is indicated by the multiplicity of their absorption and fluorescence maxima. Measuring spectra at low temperatures gives a possibility to observe the distinct bands separately, however, some of the bands are still strongly overlapped. Derivative spectroscopy is widely used as well but the overlap of derivation sidebands hinders the resolution. The deconvolution of spectra into Gaussian components is a sophisticated method but its results are often discussed because of the subjectivity in the definition of the number of components. In this work, we studied leaves and different sections of epicotyls of dark-grown pea seedlings of various ages and developmental stages. 77 K spectra of 100 samples were recorded for each tissue regions. The spectra were smoothed, corrected for their baselines and for the wavelength dependent sensitivity variations of the spectrofluorometer. The total integral values were calculated and the spectra were normalized for these values. The spectra were loaded into a worksheet of Excel and AVERAGE (arithmetic mean function) and AVEDEV (average of the absolute deviations of data point of their mean function) were calculated for each amplitude value of the spectra. The AVERAGE spectra of the different sample groups hardly differed from each other, but even the strongly overlapped emission bands sharply separated in the AVEDEV spectra. This way the emission bands of the 629, 636, 655 nm protochlorophyllide forms were clearly distinguished without any further calculations. The AVEDEV spectra were then calculated for various data groups, representing different developmental stages of the seedlings. The band amplitudes in the AVEDEV spectra varied showing biological variability of the fluorescence signals and thus the variation of the protochlorophyllide-protein complexes in the etioplasts. The calculation of AVEDEV spectra seems to be a suitable tool for studying physiological processes effecting plastid differentiation. Acknowledgement This work has been supported by the Hungarian Scientific Research Fund (OTKA T 038003).

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47. Chlorophyll bleaching by UV-irradiation in vitro and in situ: absorption and fluorescence studies ZVEZDANOVIC 1, J., CVETIC 2, T., VELJOVIC-JOVANOVIC 3 S. AND MARKOVIC 1, D. Faculty of Technology, Bulevar Oslobodjenja 124, Leskovac, 16000, Serbia, [email protected] University of Belgrade, Faculty of Biology, Takovska 43, Belgrade, 11000, Serbia 3 Centеr for Multidisciplinary Studies, University of Belgrade, Kneza Višeslava 1а, Belgrade, 11030, Serbia 1 2

Beyond being the ultimate driving force of photosynthesis and its important regulatory factor, solar light is also a major source of stress to photosynthetic organisms, with lot of accompanying consequences. Depletion of stratospheric ozone has led to increased levels of ambient UV-light (mainly UV-B light, 280320 nm) that may cause severe damage to biomolecules. Although photosynthetic pigments - chlorophylls and carotenoids - absorb in VIS region, their structure is significantly altered when exposed to UV-light [1]. As chlorophyll (Chl) is major photosynthetic pigment its UV-induced alteration may cause an impairment of photosynthetic process [2]. We investigated stability of chlorophylls in acetone and n-hexane extracts (in vitro) and in isolated thylakoids (in situ) in this work by absorption and fluorescence spectroscopy during UV-irradiation from three different ranges (UV-A, UV-B and UV-C). Chlorophyll undergoes destruction (bleaching) obeying the first-order kinetics. The bleaching kinetics is mainly governed by UV-photon energy input and the molecular organization of chlorophyll (“monomeric” Chl in acetone, “dimeric” form of Chl in hexane and highly aggregated Chl molecules within light harvesting complex in thylakoids). Products obtained after different periods of irradiation (from 2 to 180 min), characterized by fluorescence emission maximum and absorption spectra, differed depending on UV range indicating different mechanisms by UV-A and B- compared to UV-C-induced degradation.

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___ non-irradiated Chls _ _ UV-A irradiated Chls _._ UV-B irradiated Chls ..... UV-C irradiated Chls

~667 nm Fluorescence maximums of Chls

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Fig. 1. Fluorescence spectra of UVirradiated chlorophyll in acetone after ~10 min of exposure to UV radiation. Total chlorophyll concentration was 1,2x106 moldm-3. Emission spectra were recorded at room temperature, in 3 ml quartz cuvette; excitation wavelength was 430 nm; for emission in 450-750 nm range both excitation and emission slits were 1nm, while for product emission spectra (450600nm) excitation and emission spectra were set to 2 and 4 nm, respectively.

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Acknowledgements J. Zvezdanovic is a recipient of a fellowship granted by Ministry of Science and Environmental Protection Republic of Serbia. References [1] Merzlyak MN, Pogosyan SI, Lekhimena L, Zhigalova TV, Khozina IF, Cohen Z, Khrushchev SS (1996) Rus J Plant Physiol 43: 160-168. [2] Teramura, A H, Ziska, L H, (1996) In: Photosynthesis and the Environment, (Baker N, ed.), Kluwer, pp. 435-450.

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48. Interaction of Porphyrin Derivatives with Liposomes Containing Unsaturated Lipid VOSZKA 1, I., GRÓF 1, P., CORRAD I2, G., MAILLARD 3, P., STEINHOFF 4, H-J. AND CSÍK 1, G. Institute of Biophysics and Radiation Biology, Semmelweis University, Puskin str. 9., Budapest, 1088, Hungary, [email protected] 2 Research Institute for Solid State Physics and Optics, Hungarian Academy of Sciences, H-1525, Budapest, P.O.B. 49, Hungary 3 Institute Curie, Section de Biologie, Orsay, France 4 Fachbereich Physik, Universität Osnabrück, Germany 1

Photodynamic treatment is a combination of visible light and light-absorbing chemicals. It is believed to be mediated by the generation of singlet oxygen. Due to the short half-life and short diffusion path of this species the primary damage is expressed in the immediate vicinity of the photosensitizer. Hydrophobic photosensitizers are known to be localized in cellular and mitochondrial membranes serving as the main targets of photodynamic action. Therefore the efficiency of the photodynamic process crucially depends on the pattern of dye localization in the membranes and the factors influencing the photosensitizer–membrane interactions. We examined the effect of molecular structure of porphyrin derivatives on their localization in the membrane structure, and the effect of photosensitizer on the lipid structure. For our studies porphyrin derivatives with symmetrical and asymmetrical structure were selected. Liposomes containing unsaturated lipids and spin labels were used to study the interaction. The localization of liposome-bound dyes was studied by electron paramagnetic resonance (EPR) spectroscopy. The efficiency of photodynamic treatment was followed by the decay of the EPR signal amplitude. The porphyrin–lipid interaction strongly depends on the symmetry and polarity of porphyrin derivatives. For tetraphenyl porphyrins we propose that the asymmetrical derivative is situated much deeper within the membrane than the symmetrical one. Only unsaturated fatty acids were sensitive for the phototreatment, and porphyrins localized in closer connection with the double bond of the unsaturated fatty acid were more effective[1]. Acknowledgement This work was supported by Grants ETT 083/2003 from the Ministry of Health of Hungary and by Hungarian Grant OTKA K-60086. References [1] Voszka, I., Budai, M., Szabó, Zs., Maillard, P., Csík, G. and Gróf, P.(2007) Chem. Phys. Lipids 145: 63-71.

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49. UV-effects on antioxidant capacities of selected carotenoids in the presence of lecithin CVETKOVIC, D., AND MARKOVIC, D. Faculty of Technology, Bulevar oslobodjenja 124, Leskovac, 16000, Serbia, [email protected]

Depletion of stratospheric ozone has led to an increase of biologically damaging UV-light at ambient levels (mainly UV-B light, 280-320 nm). The induced consequences affect many crucial biologically important processes of global importance, such as DNA replication [1], photosynthesis [2] etc. Also, UVlight can generally initiate a lot of harmful free radicals mediated processes, lipid peroxidation (LP) among them. Lipid peroxidation is tightly connected with many pathological processes which finishes some form of cancer at the very end, melanoma skin cancer among them [3]. Lipid peroxidation is mostly controlled by antioxidants action in vivo. In recent years, carotenoids have received wide research interest as potential antioxidants [4]. Antioxidant action of four selected carotenoids (two carotenes, β-carotene and lycopene, and two xanthophylls, lutein and neoxanthin) on UV-induced lecithin’ lipid peroxidation has been studied by TBA and DPPH test. TBA test is based on absorbance measurements of complex formed between malondialdehyde, secondary product of lipid peroxidation, and thiobarbituric acid, at 532 nm. DPPH test is based on measurements of decreasing free DPPH radicals` absorbance at 517 nm. The antioxidant capacities of investigated carotenoids appeared to be strongly affected by UV-action from all three UV-ranges (UV-A, UV-B & UV-C, Fig. 1). High energy input of the involved UV-photons plays major governing role in supressing the studied carotenoids` antioxidant capacities. The results suggest a minor remained contribution of selected carotenoids to prevention of the UV-induced lecithin peroxidation in the studied system, partly dependent of their structures.

Fig. 1. (A) Decrease of inhibition of UV-induced lecithins peroxidation in the presence of β-carotene during increasing UV- A irradiation intervals at 350 nm – results of TBA-MDA test. (B) Decrease of DPPH radical scavenging capacity of β-carotene during increasing UV-B irradiation intervals at 300 nm – results of DPPH test. Acknowledgement D.C. is a recipient of a fellowship granted by Ministry of Science and Environmental Protection, Republic of Serbia. . References [1] Ichihashi M, Ueda M, Budiyannto A, Bito T, Oka M, Fukunaga M, Tsuru K, Horikawa T (2003) Toxicology 189: 21–39. [2] Teramura H, Ziska LH (1996), In: Photosynthesis and the Environment, Chapter 18, Kluwer Academic Publishers, pp. 436-450. [3] Ouhtit A, Ananthaswamy HN (2001) Journal of Biomedicine and Biotechnology 1:1: 5-6. [4] Burton GW, Ingold KU (1984) Science 224: 569-573.

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50. Fluorescence Lifetimes and Spectral Properties of Protochlorophyll in Organic Solvents MYŚLIWA-KURDZIEL 1, B., SOLYMOSI 1, K., KRUK 2, J., STRZAŁKA 2, K., AND BÖDDI 1, B. Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, Kraków, 30-387, Poland 2 Department of Plant Anatomy, Eötvös University, Pázmány P. s. 1/C., Budapest, 1117, Hungary, [email protected] 1

In the present study, absorption and fluorescence spectra of protochlorophyll (Pchl), as well as its fluorescence lifetime were investigated in organic solvents with different physical and chemical properties. The results allowed us to distinguish between non-specific (which depend only on bulk solvent parameters), and specific (e.g. H – bonds) solvent-solute interactions. The position of the Qy band of Pchl absorption spectrum was observed within the range of 15 873-16 064 cm-1. The energy of this band linearly decreased with the function of solvent polarity R(n2), defined as R(n2)= (n2-1)/(n2+2). Energy of fluorescence emission band decreased for increasing solvent orientation polarizability, which is a function of both the dielectric constant (ε) and the refractive index (n). The values of the Stokes shift varied beween 38 (in hexane) and 345 cm-1 (in methanol); in the case of other solvents, these values varied between the limits above. An increase of this parameter was observed for increasing solvent orientation polarizability, however, the extent of this increase reflected the presence of specific solvent-Pchl interaction. The fluorescence decay of Pchl was monoexponential in case of all investigated solvents. In general, the fluorescence lifetime of Pchl decreased for increasing solvent orientation polarizability. The longest values were found for 1,4-dioxane (5.4 ns) and diethyl ether (5.2 ns). In protic solvents (e.g. primary alcohols), the Pchl fluorescence lifetime was significantly shortened and the lowest value was measured in methanol (3.5 ns). The present results are compared to respective data measured for protochlorophyllide in organic solvents that have already been published [1]. This is the first time when a complex analysis and comparison of fluorescence emission and fluorescence lifetimes of purified Pchl and protochlorophyllide was done. Our results indicate that the presence of phytol chain on the porphyrine ring influences the spectral properties of the whole chromophore. Acknowledgement This work was supported by the Polish Committee for Scientific Research grant No. 158/E-338/SPUB-M/5 PR UE/DZ 9/2001-2003, by the Hungarian Scientific Research Fund (OTKA), grant NO. T038003 and by the European Union (contract No. BIER ICA1-CT-2000-70012). References [1] Myśliwa-Kurdziel B, Kruk J, Strzałka K (2004) Photochem Photobiol 79: 62-67.

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51. Phototransformation of Protochlorophyllide Forms under Selective Laser Light Illumination KÓSA, A. AND BÖDDI, B. Department of Plant Anantomy , Institute of Biology, Eötvös University , Pázmány P. s. 1/C, Budapest H-1117, Hungary, [email protected]

The activity of NADPH:protochlorophyllide oxidoreductase (POR) has a key regulatory function in chlorophyll biosynthesis. In higher plants, this enzyme is light-activated. The POR units form ternary complexes with NADPH and protochlorophyllide ?→ →? C676 (Pchlide). These ternary complexes are either P633 monomers or dimers and oligomers built into the ↓↑ etioplast inner membranes. Kinetical measurements P644 → C683 suggested dynamic interconversions among the above↓ ↑ mentioned complexes. Fig. 1. summarizes the main → C692 pathways of phototransformation and interconversion P655-657 processes of protochlorophyllide complexes in wheat leaves. The absorption maxima of monomers, dimers Pxxx: Pchlide complex emitting at xxx nm and oligomers are at 628-633, 638 and 650 nm, Cxxx: Chlide complex emitting at xxx nm respectively. Thus the 632.8 and 650 nm laser lines directly excite the monomers and the oligomers. The question can be raised which forms transform into Fig. 1. Main pathways of interconversions and photoreducetion of protochlorophyllide forms. chlorophyllide (Chlide) with separate or combined illumination at equal photon flux densities (PFD). In this work dark-grown wheat leaves as well as isolated and purified etioplast inner membrane fractions, i.e. prolamellar body- and prothylakoid-enriched preparations were studied. The samples were illuminated with 632.8 and/or 650 nm laser light of various PFDs for different time periods and were frozen into liquid nitrogen. 77K fluorescence emission spectra were recorded. Both laser lights caused the formation of shorter-wavelength emitting Chlide forms at low light intensities and/or short-time illumination than at higher intensities and/or longer periods. This can be explained by the different localization of Pchlide complexes in the membranes. Despite of the selective excitation, high PFD 632.8 nm light caused the production of C692 as the 650 nm light itself. These results prove a very effective energy migration among the Pchlide forms as well as very fast interconversions among the different pigment-protein complexes in the native etioplast inner membranes. Acknowledgements This work has been supported by the Hungarian Scientific Research Fund (OTKA T038003). The authors are grateful to Christer Sundqvist (Göteborg University) for providing the etioplast inner membrane preparations and for the valuable discussions of the results. Thanks for the Department of Biophysics and Radiation Biology, Semmelweis University for lending He-Ne laser for the experiments.

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52. Fluorescence Lifetimes of Distinct Protochlorophyll Forms with Similar Steadystate Fluorescence Characteristics in Triton X-100 Micelles SOLYMOSI 1, K., MYŚLIWA-KURDZIEL 2, B., KRUK 2, J., BÖDDI 1, B., AND STRZAŁKA 2, K. 1 2

Department of Plant Anatomy, Eötvös University, Pázmány P. s. 1/C., Budapest, 1117, Hungary, [email protected] Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, Kraków, 30-387, Poland

Protochlorophyllide (Pchlide) is a chlorophyll biosynthesis precursor that accumulates in dark-germinated angiosperm plants. Protochlorophyll (Pchl), i.e. Pchlide ester is a side product of this biosynthesis pathway; it is usually present in minute amounts in the leaves of dark-grown seedlings but is accumulated, even in the crystallized form in seed coats of some pumpkin species. Pchl and Pchlide pigments are present in different molecular microenvironments within the plants and, as a consequence, they have different spectral properties. The characterization of the fluorescence lifetimes of the different forms is very complicated in vivo because of the strongly overlapping bands, energy migration among them and the photoreduction of enzyme-bound Pchlide molecules, which is triggered by the measuring light at room temperature [1]. Earlier data have shown that Pchlide and Pchl have similar steady state fluorescence properties and Pchl was widely used to model the in vivo Pchlide forms in artificial model systems such as micellar solutions [2]. However, the question can be raised if the geometrical differences between Pchlide and Pchl complexes influence the fluorescence lifetime properties. Therefore, we investigated the steady state and time-resolved fluorescence characteristics of Pchl dissolved in neat Triton X-100 and in Triton X-100 micelles [3]. Varying the concentration of Pchl or diluting the micellar solutions either with buffer or with micellar solution, 631-634, 645-655, 680-692 and above 700 nm emitting Pchl complexes were prepared, the ratios of which varied among each other depending on the used concentrations and the way of sample preparation. The fluorescence decay of the 631634 nm emitting (monomeric) form had mono-exponential character with 5.4-ns fluorescence lifetime. This form resembles the spectral properties of monomeric Pchlide forms found in dark-grown seedlings that have similar, even slightly higher lifetime values in the etioplast inner membranes [1, 4]. The long-wavelength Pchl complexes (aggregates) had two fluorescence lifetime values in the range of 1.4 - 3.9 ns and 0.15 - 0.84 ns, which showed high variability in different environments. Mono- or double-exponential fluorescence decay was found for the fluorescence band at 680-685 nm. The data of this work show that despite their very similar steady-state fluorescence properties, Pchl complexes can differ in their fluorescence lifetimes that may reflect different molecular structures, intrinsic geometries or different molecular interactions [3]. This underlines the importance of complex spectroscopic analyses for the precise description of native and artificial chlorophyllous pigment forms. Acknowledgement This work was supported by the Polish Committee for Scientific Research grant No. 158/E-338/SPUB-M/5 PR UE/DZ 9/2001-2003, by the Hungarian Scientific Research Fund (OTKA), grant NO. T038003 and by the European Union (contract No. BIER ICA1-CT-2000-70012). References [1] Myśliwa-Kurdziel B, Franck F, Strzałka K (1999) Photochem Photobiol 70: 616-623. [2] Böddi B, Kovacs K, Láng F (1983) Biochim Biophys Acta 722: 320-326. [3] Myśliwa-Kurdziel B, Solymosi K, Kruk J, Böddi B, Strzałka K (2007) J Photochem Photobiol B:Biol 96: 262-271. [4] Myśliwa-Kurdziel B, Amirjani MR, Strzałka K, Sundqvist C (2003) Photochem Photobiol 78: 205-215.

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53. Role of fructose in the adaptation of plants to cold-induced oxidative stress BOGDANOVIĆ 1, J., MOJOVIĆ 2, M., MILOSAVIĆ 3, N., MITROVIĆ 1, A., VUČINIĆ 1, Ž., AND SPASOJEVIĆ 1, I. Center for Multidisciplinary Studies, Kneza Višeslava 1, Belgrade, 11000, Serbia, [email protected] Faculty for Physical Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, 11000, Serbia 3 Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, 11000, Serbia 1 2

Role of soluble sugars, such as glucose and fructose, in the redox metabolism is still an unresolved issue. While, the increased level of glucose in cells, is associated with the production of ROS though glucose autooxidation, glucose feeding of the oxidative pentose-phosphate (OPP) pathway can enhance NADPH and fructose production [1]. Fructose has been speculated to poses high ROS scavenging capacity [2], as we have observed recently [3]. Since, the exposure of plants to low temperatures is known to lead to both, the oxidative stress and changes in the metabolism of sugars [4], we investigated potential correlation between misbalanced oxidative status and the levels of fructose and glucose, in plants exposed to low temperatures. Whole pea plants (Pisum sativum L.) were exposed for 3 hours to temperature of 2oC and to irradiance of 70 µmol×m-2×s-1. Leaves were harvested periodically and measurements of oxidative status and sugar levels, using EPR spectroscopy, biochemical assays, and HPLC, were performed. Low temperature combined with moderate light led to the accumulation of oxidized form of PSI pigment P700 (P700+), which represents an EPR active species [5], during the first two hours of treatment. This shows that under these experimental conditions photoinhibition and oxidative stress were provoked. However, after the second hour of treatment, the level of P700+ rapidly decreased, indicating that the plant have “activated” antioxidative response. ABTS assay showed that the antioxidative status in plant leaves raised during the second hour of treatment, prior to the decrease of the level of P700+. Such accommodation to cold-induced oxidative stress was preceded by the significant increase of the level of fructose. The level of glucose has only slightly increased, so the principal cause of the increase of the level of fructose could be the increase of glucose feeding of OPP pathway. Since, fructose and its phosphorylated derivatives showed to be effective antioxidants, fructose could play a major role in the accommodation of plants to oxidative stress and the development of resistance to photoinhibition [5]. Acknowledgement This work was supported by the Grant from the Ministry of Science and Environmental Protection of Republic of Serbia (B143016). References [1] Couée I, Sulmon C, Gouesbet G, El Amrani A (2006) J Exp Bot 57: 449-459. [2] Girard A et al. (2005) Nutrition 21: 240-248. [3] Maksimović V, Mojović M, Vučinić Ž (2006) Carbohydr Res 341:2360-2369. [4] Streb P, Aubert S, Gout E, Bligny R (2003) Physiol Plantarum 118: 96-104. [5] Ivanov AG, Morgan RM, Gray GR, Velitchova MY, Huner NPA (1998) FEBS Lett 430: 288-292.

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54. Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation ZUPÁN 1, K., EGYEKI 1, M., TÓTH 2, K., FEKETE 1, A., HERÉNYI 1, L., MÓDOS 1, K., CSÍK 1, G. 1 2

Institute of Biophysics and Radiation Biology, Semmelweis Univ., Puskin 9., Budapest, H-1088 Hungary, [email protected] Biophysik der Makromoleküle, DKFZ, Heidelberg, Germany

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to removal of non-enveloped viruses from aqueous media [1]. Cationic porphyrins have long been of interest because of their binding interactions observed with DNA [2]. Our recent results [3,4] proved that the binding modes of TMPyP in the NP do not include porphyrinprotein binding but TMPyP binds to the DNA part of the NP by two distinct binding modes, i.e., external binding and intercalation. The spectroscopic methods used in our recent publications also facilitate quantifying bound species and free porphyrin under various experimental conditions. Here we tested the virus inactivation capability of meso-Tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4-N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. Beyond the evaluation of virus inactivation efficiency of two cationic porphyrins, we address two questions here. (I) Does the specific DNA binding of porphyrin lead to specific DNA damages in the nucleoprotein complex? (II) Is bound porphyrin more effective in the photosensitized virus inactivation than the free species? T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be an efficient photosensitizer of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding. Acknowledgement This work was been supported by research grant DAAD D/03/30958 and Joint Project of DFG – Hungarian Academy of Science 436 Ung. References [1] Wagner, SJ (2002) Transfus. Med. Rev. 16:61-66. [2] McMillin, DR, Shelton, AH, Bejune, A, Fanwick, PhE, R. K. Wall, RK (2005) Cord. Chem. Rev. 249:1454-1459. [3] Zupan, K, Herenyi, L, Toth, K, Majer, Zs, Csík, G (2004) Biochemistry 43:9151-9159. [4] Zupan, K., Herenyi, L, Toth, K, Egyeki, M, Csík, G (2005) Biochemistry 44:15000-15006.

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55. On mechanism of uranium binding to cell wall of Chara Fragilis DAKOVIĆ 1, M., KOVAČEVIĆ 1, M. , ANDJUS 2, P. R. AND BAČIĆ 1, G. 1 1

Faculty of Physical Chemistry, Studentski trg 12-16., Belgrade, 11000, Serbia, [email protected] Faculty of Biology, Studentski trg 12-16., Belgrade, 11000, Serbia

Biosorption of heavy elements, especially uranium, from nuclear waste liquids and contaminated surface waters and soils has attracted special interest recently [1,2,3]. However, the detailed mechanism of uranium uptake by plants is not well understood. [4]. The aim of this work is to investigate the role of cell wall components of freshwater alga Chara fragilis in uranium localization and sequestration from its solution by methods of FTIR, VIS spectroscopy and confocal microscopy. Investigation of uranium binding by Chara Fragilis has showed that the sorption process occur in two components. Comparison of FTIR spectra of native and treated dry cells were showed a shift of carboxyl group stretching band to lower wavenumbers, which can be explained by stabilisation of its structure due to uranium binding. Uranium binding to polysaharide extract of cell wall, which resembles a pectinic structure, has also shown shift of carboxyl band in FTIR spectra. Second major change in FTIR spectra is splitting of the carbonate band (carbonates in form of calcite are normally present in Chara cell wall). Position of appeared band corresponds to that of uranyl [5] and its intensity changes with uranium concentration in solution. Incorporation of uranium in calcite seems to be more pronounced in slightly basic solutions whats in a agreement with uranyl carbonate abundance in solution at these pH values. Cross sections of treated Chara cells were also investigated with confocal microscopy in order to elucidate uranium localization within structures of cell wall. Since Chara fragilis cell wall has in great manner similar structure to that of nonvascular land plants (mosses and lichens), it can be used as a model for the uranium sequestration mechanism by these plants. References [1] Macaskie, L. E., (1991) CRC Crit. Rev. Biotechnol. 11: 41-112. [2] Volesky, B., Holan Z. R. (1995) Biotechnol. Prog. 11: 235-250. [3] Guibal, E. , Roulph, C, Le Cloirec, P (1992) Water Res., 26: 1139-1145. [4 ] Fourest, E., Volesky, B. (1996) Environ. Sci. Technol. 30: 277-282. [5] Shurmel, L. B., Shatalov, V. V. et al., Atomic Energy, 90:218-223

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56. Characterization of blood clot structure by MR microscopy VIDMAR 1, J., BLINC 2, A., AND SERŠA 1 I. 1 2

Jožef Stefan Institute, Jamova 39, Ljubljana, 1000, Slovenia, [email protected] Department of Vascular Diseases, University Medical Center , Zaloška 7, Ljubljana, 1000, Slovenia

Several attempts have been made to link MRI characteristics of blood clots to their susceptibility to thrombolytic treatment. High resolution T1-weighted MRI uncovers additional information about the morphology of blood clots [1]. Platelets-rich clots and platelets-rich areas in the clots are less susceptible to thrombolysis than platelets-poor clots and platelets-poor areas [2]. Our experiments showed that T1weighted MRI of pulmonary emboli taken ex vivo was capable of non-invasive assessment of the red-rich and platelet-rich components. Furthermore, we found out that platelets aggregates display a brighter signal in T1-weighted MRI versus erythrocyte part, which is somewhat surprising since platelets do not contain paramagnetic haemoglobin that would enhance magnetic relaxation. When the fibrin network is formed, erythrocytes are not covalently bound into the net and the red cell-rich part of the clot retains abundant extracellular space filled with serum, whereas platelets bind with each other via GPIIbIIIa receptors and fibrinogen forming tight aggregates.It is therefore likely that compactly packed platelets resemble the “solid tissue” where cellular proteins enhance magnetic relaxation. We measured T1 values of sedimented packed platelets versus different concentrations of erythrocytes in plasma to explain why platelet-rich areas give more intense MRI signal than the erythrocyte part of the clot. MR spectrometry results in approximately equal values of T1 in 100% packed platelets aggregates and 60%-80% erythrocytes in plasma. T1 values of 100% packed platelets are equal to 1220 ± 11ms, of 60% erythrocytes to 1176 ± 12ms and of 80% erythrocyte to 1000 ± 8ms. Thus, platelet aggregates shorten the T1 of water to an equal degree as retracted red cell clots with a hematocrit of 60-80%, assuming that the effect of fibrin network may be neglected. Along detecting morphology of the clot, another characteristic of blood clot, i.e., clot retraction can be assessed by MRI. Assessment of the clot retraction is important for estimation of the clot lysability. Clot retraction strongly changes the MRI appearance of clots and decreases their lysability by thrombolytic agents [3,4]. Our MR images of artificial clots showed that retraction occurred in the first hours after the clot formation but it was unlikely to influence the structure of older thrombi. Based on our preliminary results, we conclude that MRI is capable not only of non-invasive assessment of clot morphology, but could be also used to assess clot retraction or compaction. Results of this study could have an important role in clinical trials for choosing appropriate and effective thrombolytics. Acknowledgement We thank dr. Dragoslav Domanovic from Blood Transfusion Centre of the Republic of Slovenia for providing human plasma and platelet concentrate. References [1] Tratar, G, Blinc, A, Podbregar, M, Kralj, E, Balažic, J, Šabovič, M, Serša, I (2007) Characterization of pulmonary emboli ex vivo by magnetic resonance imaging and ultrasound Thromb.Research: [Epub ahead of print]. [2] Stump, DC, Mann, KG, Mechanisms of thrombus formation and lysis (1988) Ann Emerg Med 17(11):1138-47. [3] Blinc, A, Keber, D, Lahajnar, G, Zupančič, I, Zorec-Karlovšek, M, Demšar, F Magnetic resonance imaging of retracted and nonretracted blood clots during fibrinolysis in vitro (1992) Haemost. 22:195-201. [4] Kozak, M, Mikac, U, Blinc, A, Serša, I (2005) In:Lysability of arterial thrombi assessed by magnetic resonance imaging, Vol. 34, VASA (Creutzig A, Enzler M, Mahler F, Partsch H, eds.), pp. 262-265.

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57. Detection of changes in mice metabolism after X-ray irradiation by 31P NMR spectroscopy ŠENTJURC 1, M., KRANJC 2, S., SERŠA 2, G., SEPE 1, A., VIDMAR 1, J., SERŠA 1, I. 1 2

Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Department of Experimental Oncology, Institute of Oncology, Zaloška 2, 1000 Ljubljana, Slovenia

The aim of this study was to evaluate the potential application of phosphorous NMR spectroscopy to determine prior exposure to ionizing irradiation. The received dose of radiation cannot be accurately determined unless the subject is equipped at the time of exposure with a dosimeter. Alternatively, the effects of irradiation can be determined indirectly by the damage the irradiation caused to the genetic material [1] and the changes in metabolism [2]. The latter was measured by phosphorous NMR spectroscopy. For the study three groups of mice were selected. The first and the second group were exposed to 3.5 Gy and 7 Gy of x-ray irradiation respectively while the third group was a control group and did not receive any irradiation. Metabolic changes in all mice groups were measured using 31P spectroscopy at different time points after irradiation. No significant change in metabolism was observed in either the control group or the 3.5 Gy group at all times after irradiation while a significant difference was observed between the control group and the 7 Gy group 2 days (Fig. 1) or more after irradiation. The irradiated group has in 31P NMR spectra a significantly lower phosphocreatine peak (PCr), which represents energy supply for muscles. The decrease of PCr peak can thus be used as a biological sensor for the received dose of irradiation if the dose is close to the fatal dose. 1,7

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Fig. 1: Changes in the ATP/PCr peak ratio normalized to the first measurement as a function of time after irradiation for: 3.5 Gy group (a) and 7 Gy group (b). References [1] Hlinkova, E., Bobak, M (2004) J. Appl. Biomed. 2: 101-109. [2] Ng, C.E., McGovern, K.A., et al. (1992) Magn. Reson. Med. 27: 296-309.

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58. The Effect of Flow Separation and its Influence to Permeation of Non-occlusive Blood Clots with Thrombolytic Agent During Thrombolytic Therapy GROBELNIK 1, B., VIDMAR 1, J., TRATAR 2, G., BLINC 2, A. AND SERŠA 1, I. 1

Jožef Stefan Institute, Jamova 39, Ljubljana 1000, Slovenia of Vascular Diseases, University of Ljubljana Medical Centre, Ljubljana 1000, Slovenia

2 Department

Thrombolytic therapy aims to dissolve blood clots and restore vessel patency. It is used in treatment of ischemic stroke [1], pulmonary embolism and acute arterial thrombosis. Thrombolysis starts with activation of the proenzyme plasminogen into the active serine protease plasmin [2] that is very efficient in degrading fibrin network. Activation of plasminogen is achieved by adding a thrombolytic agent into the circulation. Recombinant tissue type plasminogen activator (rt-PA) is widely used fibrin-specific thrombolytic agent that excels in ability to activate only the plasminogen in contact to the fibrin network. One of the major problems in thrombolysis is efficient and fast transport of the thrombolytic agent into the clot. Diffusion, as a mean of thrombolytic agent transporation is very slow [3]; for this reason is the dissolution of occlusive blood clots slow and inefficient. The dissolution is much faster in non-oclusive blood clots having a flow channel with established fast axially directed blood flow. The dissolution is then faster because of two reasons: firstly, thrombolytic agent is delivered to the clot by the flow convection, which is significantly faster process than diffusion, and secondly, the blood flow exerts large mechanical forces to the surface of the clot that act is parallel with the biochemical processes and help degrading the clot more efficiently. Our recent results of dynamical 3D magnetic resonance imaging (MRI) of blood clot dissolution in the artificial circulation system showed that non-occlusive blood clots are dissolving nonuniformly. The dissolution is considerably faster at the entrance of the flow channel than further downstream. The effect is considerable to the entrance distance of approximately one tenth of the entrance length, which is according to the literature [4] equal to 0.06 Re d (Re is the Reynolds number of the flow, and d is the vessel diameter). The faster dissolution may be assigned to the higher shear velocity at the entrance and therefore larger viscous forces to the clot surface. However, our recent results presented in this work indicate, that the dissolution is faster also due to better permeation of the clot with the thrombolyic agent. This effect was studied by dynamical MRI of clots in the artificial circulation. The MRI contrast agent Gd-DTPA, which was used as a surrogate for the thrombolytic agent, was added to the plasma in the circulation system and saturation of the clot as function of time was measured at different flow rates. The results confirmed our expectations that permeation of the thrombolytic agent into the clot is faster at the entrance of the flow channel than further downstream. Figure 1 shows permeation of the MRI contrast agent into a clot. The clot is imaged in the sagital slice, the flow channel is down the clot and the flow direction is from the right to the left. The part of the clot before permeation of the contrast agent appears dark in MR images while the part of the clot with the contrast agent is bright. This result, which is a consequence of pressure distribution along the clot and its porous structure, was obtained also in computer simulation of the Figure 1 permeation process. References [1] Del Zoppo, (1999) Thromb Haemost 82: 938-946. [2] Collen, D (1999) Thromb Haemost 82: 259-270. [3] Blinc, A, Francis, CW (1996) Thromb Haemost 76: 481-491. [4] Nichols, WW, O'Rourke, MF (2005) In: McDonald's Blood Flow in Arteries: Theoretical, Experimental and Clinical Principles, Fifth Edition (London: Hodder Arnold), pp. 38-39.

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59. Visualization of activated GTPase Rac1A in living Dictyostelium cells FILIĆ V. 1, FAIX J. 2, AND WEBER I. 1 1 2

Ruñer Bošković Institute, Dept. Molecular Biology, Bijenička 54, HR-10000 Zagreb, Croatia, [email protected] Hannover Medical School, Institute for Biophysical Chemistry, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany

In pursuit of visualizing the activated form of the small GTPase Rac1A in living Dictyostelium cells, a fusion between CRIB domain of a Pak1 kinase and the yellow fluorescent protein (YFP) was constructed. Pak1-CRIB, or GBD for GTPase-binding domain, is known to bind specifically to activated, GTP-bound form of Rac GTPases [1]. This construct, Pak1-CRIB-YFP, was expressed in wild-type Dictyostelium cells and its localization was monitored by laser scanning confocal microscopy in the course of cell migration, endocytosis and cytokinesis. During migration, Pak1-CRIB-YFP localizes to leading edges and pseudopod tips of moving vegetative and aggregation-competent cells. During phagocytosis and macropinocytosis, Pak1-CRIB-YFP localizes to phagocytic cups and crown-like cortical extensions that mediate uptake of fluid in Dictyostelium. Surprisingly, however, no enrichment of the construct was detected during mitotic cell division, neither at the cell poles, nor at the cleavage furrow. These results are in a sharp contrast with the localization of another Rac1A-binding protein DGAP1, an IQGAP-related protein which is enriched at the trailing end of migrating cells and at the cleavage furrow of dividing cells [2]. Taken together, these findings indicate that small GTPase Rac1A is engaged in regulation of divergent signaling pathways leading to formation of cortical domains with different localizations and functional roles. References [1] Sander, EE, van Delft, S, ten Klooster, JP, Reid, T, van der Kammen, RA, Michiels, F, Collard, JG (1998) J Cell Biol 43: 1385-1398. [2] Faix, J, Weber, I, Mintert, U, Kohler, J, Lottspeich, F, Marriott, G (2001) EMBO J 20: 3705-3715.

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60. Impedance Parameters of Apple Tissue from Peel to Core VOZÁRY, E. Corvinus University of Budapest, Somlói Street 14-16., Budapest, H-1118, Hungary, [email protected]

The parameters of impedance spectrum measured on vegetables and fruits can be used in quality assessment [1,2]. The impedance spectrum of plant tissue in low frequency range – from 10 Hz till 1 MHz – can inform about the state of cells [3]. The impedance parameters have long been used for determination moisture content in fruit tissues and in grains during drying [4,5]. For use of impedance parameters in quality assessment it is necessary to know the relationship of biological structure with impedance parameters. The aim of this work was to evaluate the impedance parameters of apple tissues placed at various parts in whole apple. Impedance spectrum of apple tissues placed at various distances under the pill was determined in Jonathan, Idared and Gála apples purchased at local market. Two different two-electrode configuration were applied: a.) ECG (electrocardiograph) electrodes from Fiab Spa with contact surface of 10 mm diameter, b.) copper pin electrodes covered by gold. The magnitude and phase angle of impedance were measured with a HP 4284A precision LCR meter in frequency range from 30 Hz till 10 MHz at 1 V voltage. The stray admittance and the residual impedance from measured spectra were eliminated by an open-short correction. Each corrected spectrum was approached by a model consisting of a serial connection of a resistor, R1, and a distributed Cole-element, R/(1+ (iτω )ψ ), where R is the distance between the two intersection of Cole diagram, τ is a relaxation time, ψ is an exponent characterizing the distribution of relaxation times, ω = 2πf , f is the measuring frequency and i is the imaginary unit [3]. The resistance of apoplasm and symplasm can be evaluated from the intersection points of circular arc with the real axis [3]. The changes of impedance parameters in the function of distances under peel were the same for both experimental serials realized with pin and ECG electrodes, respectively. The magnitude of impedance decreased as the distance from the peel increased, the impedance magnitude of apple tissue at core was the half of impedance magnitude of apple tissue under peel. The minimum of phase angle curve shifted toward the higher frequencies as the place of measurement came nearer to apple core. The resistance of both apoplasm and symplasm decreased from the peel till core. The tendency of observed changes was the same for all three investigated apple varieties. The decrease in impedance magnitude at core can be explained by that the size of cells around the core is larger than under the peel [6]. In conclusion the values of impedance parameters are sensitive to structure of biological tissues. If we use the impedance parameters in quality assessment it is necessary to let know the exact place, where these parameters were determined. Acknowledgement This work was supported by Hungarian Research Fund (OTKA, grant T 042911). References [1] Harker, FR, Maindonald, JH (1994) Plant Physiol 106: 165-171. [2] Felföldi, J, László, P, Barabássy, S, Farkas, J (1993) Radiat Phys Chem 41: 771-780. [3] Grimnes, S, Martinsen, OG (2000) Bioimpedance and Bioelectricity Basics, Academic Press New York [4] Vozáry, E, Horváth, E (1998) Control Application in Post-Harvest and Processing Technology, pp. 139-141. [5] Gillay, B, Funk, D (2005) Progr Agric Eng Sci 1: 77-93. [6] Mohsenin, NN (1986) Physical Properties of Plant and Agricultural Material, Gordon and Breach New York, London

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61. Catalase-like activity of horseradish peroxidase: EPR study of free radical production POPOVIĆ-BIJELIĆ, A.1, MOJOVIĆ, M.1, SPASOJEVIĆ, I.2 1 2

Faculty of Physical Chemistry,University of Belgrade, Studentski trg 12-16, Belgrade, 11000, Serbia Center for Multidisciplinary Studies, Kneza Višeslava 1, Belgrade, 11000, Serbia, [email protected]

We present results, obtained using EPR spin-trapping spectroscopy, of free radical production in the reaction of horseradish peroxidase (HRP) with hydrogen peroxide as the sole substrate. EPR spectra were obtained for different times of incubation and ratios of initial concentrations of reactants (R=[H2O2]0/[HRP]0). Results imply that two reactive oxygen species (ROS) were produced during the reaction: hydroxyl (
OH) and superoxide (
O2-) radicals [1]. During first 30 minutes of the reaction, the hydroxyl radical production is exponentially decreased for R < 400, hyperbolically increased for R = 400 and shows rapid increase followed by slow decrease for R ≥ 1300. It was assumed that different kinetic profiles are due to the fact that these three different H2O2/HRP ratios activate different pathways. The reaction mechanism of HRP and hydrogen peroxide comprises three catalytic pathways: catalase-like pathway, compound III pathway and the enzyme inactivation pathway [2,3]. To elucidate these results, the reaction was monitored in time for different ratios R by UV/VIS spectrophotometry. It showed that for R < 400, enzyme intermediate compound III is produced and over time it is converted back to the native form of the enzyme. For 400 < R < 1100, compound III is formed, but also some part of it is converted into the inactive form of the enzyme, pigment P-670. For R = 1100, HRP is completely deactivated, i.e. the residual activity of HRP is zero. For ratios greater than 1100 the inactivation pathway prevails. Presented results show that the ratio of the initial concentrations of reactants, R, decides which pathway will predominate in the activity of HRP. Acknowledgement This work was supported in part by Grants from the Ministry of Science and Environmental Protection of Republic of Serbia (B143016). References [1] Bacic, G. & Mojovic, M., (2005) Ann. N. Y. Acad. Sci. 1048: 230-243. [2] Arnao, M.B., Acosta, M., Del Rio, J.A., Varon, R. & Garcia-Canovas, F., (1990) Biochim. Biophys. Acta 1041: 4347. [3] Hernandez-Ruiz, J., Arnao, M.B., Hiner, A.N.P., Garcia-Canovas, F. & Acosta, M. (2001) Biochem. J. 354: 107114.

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62. Mathematical model of aspirin intolerance in asthma ANDREJ DOBOVIŠEK 1,2, BORIS ONIŠAK 5 , ALEŠ FAJMUT 2,4, UROŠ POTOČNIK 1,3 AND MILAN BRUMEN 1,2,4 University of Maribor: 1Center for Human Molecular Genetics and Pharmacogenomics, Medical faculty, [email protected] 2Department of Physics, Faculty of natural sciences and mathematics 3Laboratory for Biochemistry, Molecular Biology and Genomics, Faculty for Chemistry and Chemical Engeneering, Slomškov trg 16, 2000 Maribor, Slovenia, 4 Institute Jožef Stefan, Jamova 39, 1000 Ljubljana, Slovenia; 5 ENT Department, General Hospital Murska Sobota,

In present contribution a mathematical model of aspirin intolerance in asthma (AI) is introduced. AI is known as an allergic response of asthmatic patients on the consumption of non steroid anti-inflammatory drugs (NSAID), which leads to the impairment of asthma. According to the widely accepted theory asthma is related to higher concentrations of cystenil – leukotrienes (Cys-LT) like leukotriene C4 (LTC4) and leukotriene D4 (LTD4) in airway. LTC4 and LTD4 are known as the main inflammatory mediators metabolized from arachidonic acid (AA) and are directly involved in occurrence of asthmatic symptoms. The metabolism of AA is governed at least by two enzymes: ciklooxygenase (COX) and 5-lipooxygenase (5-LO). In the ciklooxygenase metabolic pathway enzymes COX1 and COX2 catalyzes conversion of AA into prostanglandin H2 (PGH2) which is further metabolized into prostanglandin E2 (PGE2), prostanglandin F2 – alpha (PGF2α) and short - lived intermediates: prostacyclin (PGI2) and thromboxane A2 (TXA2). In the lipooxygenase metabolic pathway AA is metabolized into leukotriene A4 (LTA4), leukotriene B4 (LTB4) and into cystenil – leukotrienes LTC4, LTD4 and LTE4. Due to lower activity of COX2 in the airway smooth muscle cells of asthmatic subjects, lipooxygenase methabolic pathway predominate over ciklooxygenase pathway – in this way LTC4, LTD4 and LTE4 are reproduced in higher concentrations. It has been clinically observed that the consumption of NSAID (drugs such as aspirin or ibuprofen) in some cases lead to an additional impairment of asthma. This phenomenon is known as aspirin intolerance (AI). Experimental data show that NSAID causes an additional inhibition of the COX2 activity, which results in lower production of PGE2. This increases the activity of LTC4 synthase by negative inhibition effect of PGE2 on LTA4 synthase activity, which results in higher production of Cys-LT. Our mathematical model is based on the above - described metabolism of AA. The model consists of five algebraic equations describing the equilibrium conditions of model variables [AA], [PGH2], [PGE2], [LTC4] and [LTA4]. Additionally, negative inhibition effect of PGE2 on LTA4 synthase activity is considered in the model. By changing the values of the parameters that simulate the activity of COX2 enzyme as well as the PGE2 inhibition effect, our model is able to predict lower values of [PGE2] and higher values of [LTC4] and [LTD4] in the metabolism of AA. The importants of different pathways in the metabolic network scheme described above is analyzed in terms of the metabolic control theory. We wish to stress, that the metabolism of AA including the effects of AI has not been considered in a unique and general mathematical model so far. From this standpoint this contribution could be viewed as a step further in the theoretical research of asthma.

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63. On kinetic modelling and large scale structure of metabolic networks BRUCK 1, J., EBENHÖH 1,2, O. AND KLIPP 1,3, E. Chair of Theoretical Biophysics, Humboldt University Berlin, Invalidenstr. 42, Berlin, D-10115, Germany, [email protected] Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, Potsdam-Golm, D-14476, Germany 3 Max Planck Institute of Molecular Genetics, Boltzmannstr. 12, Berlin, D-14195, Germany 1 2

Two studies on metabolic networks of complementary character will be presented on the poster. We conducted a large scale analysis of the chemical reaction network of the whole metabolism based on purely structural information retrieved from a genome-wide database (i). An ongoing study intends to predict the dynamics of a smaller part of yeast metabolism connecting experimental data with a detailed ODE-based model (ii). (i) This large scale structural analysis of metabolic networks focuses on neighbourhood relationships between individual reactions. We define two reactions to be neighboured if one of them provides the necessary set of substances for the other to proceed. A method is developed which allows determining all possible neighbourhood relationships categorized as 'interaction patterns'. These patterns differ in the types of participating reactions and in the way they share their reactants. The method is applied to a set of 4795 metabolic reactions contained in the KEGG database. [1] Using the above neighbourhood relationships as a concept of adjacency allows to represent the metabolic network as a directed graph in which a node represents a biochemical reaction and an edge indicates that a reaction allows another one to proceed. For this graph we calculated the distributions of node degrees, edge centralities, and strongly connected component sizes. We found that among these the distributions, that of the outgoing degrees (ie. the number of reactions a reaction provides substrates for) shows a unique characteristic. The form of this distribution does not resemble the usual power law but exhibits an exponential cutoff. Also, the effect of randomisation on the global network structure is studied. While the general approach to represent a set of biochemical reactions as a graph is not new, we introduced the above described novel adjacency concept to capture aspects of the cooperation of reactions by providing each other with substrates. (ii) Based on experimental data [2] monitoring the changes in the central carbon metabolism of yeast which occur after reducing the oxygen level in the culture, we test a differential equation based mathematical model of this system. The main goal is to predict the changes of metabolic concentrations caused by transcriptional regulation (creation of enzymes) catalysing these reactions. To this end concentrations of 17 metabolites and transcription activities (messenger RNA levels) of 69 genes coding such enzymes were measured. (work in progress) Acknowledgement The authors thankfully acknowledge the help and contribution of Reinhardt Heinrich who passed away unexpectedly on the 23rd of October 2006. József Bruck acknowledges the Marie Curie EST project "Systems Biology" (EC contract number MEST-2-CT-2004-514169) for financial support. References [1] Bruck J, Ebenhöh O, Heinrich R (2006) Genome Inform. 17(1):208-18. [2] Wiebe MG et al.(2007) FEMS Yeast Res. (in print)

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64. Correlation properties of intrinsic heart rhythm PLATISA, M.M. AND GAL, V. Institute of Biophysics, Medical Faculty, Visegradska 26, Belgrade, 11000, Serbia, [email protected]

It has been generally accepted that the heart interbeat interval time series reflects properties of autonomic control of the heart. However, we found that under very specific conditions the intrinsic rhythm of the heart could also be revealed. We focused on correlation properties of interbeat interval (RR) time series in a broad range of physiological and pathological conditions. Using detrended fluctuation analysis (DFA) we determined the short term (α1) and the long-term (α2) scaling exponent [1]. The crossover pattern between two different regimes of correlation depends on RR interval. At the shortest RR intervals (without autonomic control), extreme physiological and pathological states are characterized by the biggest difference between scaling exponents. In this case, DFA reveals a white noise over short scales (α1 ≈ 0.5) and a strongly correlated noise over large scales (α2 ≈ 1.5). With an increase of autonomic control (longer RR intervals) the difference between α1 and α2 is decreasing and this difference disappears completely in a state of efficient autonomic control. We suppose that complexity in heart rhythm is achieved through coupling between intrinsic heart rhythm and autonomic control. We suggest that the model of stochastic resonance mechanism [2] could be applied to this system. References [1] Peng C-K, Havlin S, Stanley HE, Goldberger AL (1995) Chaos 5: 82-87. [2] Gammaitoni L, Hanggi P, Jung P, Marchesoni, F (1998) Rev Mod Phys 70: 223.

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SINGLE MOLECULE TECHNIQUES, NANOTECHNOLOGY, MICROSCOPY AND IMAGING

SINGLE MOLECULE TECHNIQUES, NANOTECHNOLOGY, MICROSCOPY & IMAGING

65. Application of magnetic particles synthesized by thermal plasma for protein purification MUSKOTÁL, A.1, GÁL, L.2, FECZKÓ, T.2,3, SZÉPVÖLGYI, J.2, VONDERVISZT, F.1 Department of Nanotechnology, University of Pannonia, Egyetem u. 10., Veszprém, 8200, Hungary, [email protected] Institute of Materials and Environmental Chemistry, Chemical Research Center, HAS, Pusztaszeri u. 59-67., Budapest, 1025, Hungary 3 Research Institute of Chemical and Process Engineering, University of Pannonia, Egyetem u. 10., Veszprém, 8200, Hungary 1 2

Recently, magnetic particles have been widely studied as they have great potential both in biotechnological and biomedical applications such as cell isolation, enzyme immobilization, protein separation, magnetic resonance imaging, drug targeting etc. Proteins can be rapidly and efficiently separated at low cost by using magnetic particles. The main drawbacks of present magnetic bio-separation methods are their low binding capacity and slow mass transfer kinetics. These characteristics can be improved by increasing the specific surface area of particles on the one hand and by improving their surface functionality on the other. However, surface functionalisation generally requires complicated post-synthesis procedures [1, 2]. Our aim was to study separation of proteins by magnetic particles synthesized in a single step without subsequent surface modifications. It is well known that proteins containing multiple histidine moieties bind strongly nickel and zinc ions. In order to take advantage of particular phenomenon, nickel-zinc ferrite spinels of different compositions were directly synthesized in a radiofrequency (RF) thermal plasma reactor. RF thermal plasma reactors exhibit plasma flames of extremely high temperature (about 104K) and very steep temperature gradients. In these conditions, micro- and nanoparticles of special physical and chemical properties can be formed. In this work nickel-zinc ferrites synthesized from different precursors in an RF thermal plasma reactor were used as magnetic particles for protein separation. The ferrite particles were collected from different parts of plasma reactor and they were characterised for particle size distribution, morphology, bulk and surface chemical composition, crystalline phase composition and saturation magnetisation. According to LDPA, the ferrites had a mean particle size of 0.5 to 20 µm depending on synthesis conditions. The powders had different bulk Ni-Zn-stoichiometry as determined by ICP-OES. The surface chemical composition was analysed by XPS. Comparison of bulk and surface compositions revealed some surface segregation of the nickel and zinc content. X-ray diffraction studies referred to formation of complex spinel ferrites. A specially designed vibrating sample magnetometer was applied for measuring saturation magnetisation. The magnetization measurements indicated remarkable saturation magnetisation that referred to ferrimagnetic particles. Over-expressed (from E. coli DE3 pLysS strain) proteins of various masses (10 kDa – 27 kDa) with N- or C-terminal polihistidine tag were purified by nickel-zinc ferrite magnetic particles of different mean particle size, chemical composition and saturation magnetization, respectively. A magnetic separation stand was used to isolate the particles with attached proteins from a crude cell lysate. Non-specifically absorbed proteins were washed out, and the target protein was eluted by 20-500 mM imidazol. The protein fractions were characterised by SDS-PAGE and immunoblotting. Binding capacities and efficiencies were determined by measuring the residual protein concentration of supernatants with a UV-VIS spectrophotometer at 280 nm. Our results demonstrate that His-tagged proteins can be purified by magnetic nickel-zinc-ferrite particles. The composition and the size of particles largely affect purification efficiency. Magnetic particles studied in this work offer a convenient way for presentation of His-tagged drugs and effector proteins in biomedical applications. References [1] Lim , Y.T., Lee, K.Y., Lee, K., Chung, B.H. (2006) Biochem Biophys Res Co 344: 926-930.

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66. Effect of antimicrobial peptide-amide, indolicidin on biological membranes SZEGLETES 1, ZS., TOMPA 2, P. AND DÉR 1, A. Inst. of Biophysics, Biological Research Center of Hungarian Academy of Sciences, Temesvári krt. 62., Szeged, H-6726 Hungary, [email protected] 2 Inst. of Enzymology, Biological Research Center of Hungarian Academy of Sciences, Karolina u. 29., Budapest, H-1113 Hungary 1

Our aim was to use atomic force microscopy as an experimental tool for the detection of interactions between biomimetic interfaces subjected to various salt solutions. The two interacting surfaces were a microfabricated tip attached to a cantilever on one side and a planar surface on the other side. The surfaces were covered by self-assembled monolayers of intrinsically unstructured proteins. The results are expected to shed light on the physical chemistry of the Hofmeister effects, and have important general implications concerning the effect of water structure on protein stability and dynamics, as well. Acknowledgement Thanks are due to Veronika Csizmók, and Edit Szıllısi for preparation of MAP2C and FRET-3 proteins, and Miklós Kellermayer for kindly providing the plasmid of FRET-3. Special thank to György Váró and Zoltán Bálint for helpful discussions. This work is supported by the Hungarian Scientific Research Foundation (OTKA K 49489) and an MTA-DFG research project.

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67. Developing an ATP Driven Biomechanical Micro device BADRI 1, A.L., KELEMEN 1, L., DIEZ 2, M., ZIMMERMANN 2, B., PETERSEN 2, J., GRABER 2, P.. AND ORMOS 1, P. 1 2

Institute of Biophysics,Biological research centre,HAS, P.O box 521, Szeged, H-6701, Hungary,[email protected] Institute of Physical Chemistry, 23 a, Freiburg, 79104,Germany.

Single molecule is, in a sense the ultimate nanostructure. With the advent of optical tweezers and other methods for manipulating, probing and imaging single molecule, like molecular motors mechanics there is an increasing possibility for generating microdevices based on these molecules. The recognition of F1 ATPase as such a nanoscale molecular motor has opened the door for potential creation of hybrid microelectromechanical devices (MEMS). Our goal here is to produce an ATP driven flagella like artificial propeller. As an intermediate step we present here the binding of F1 ATPase to Ni- NTA surface and to plane glass chamber and report a successful test of rotating ATPase-bound Streptavidin-coated beads due to ATP hydrolysis. A two photon polymerisation technique was used to create 2-3 um long SU-8 rods (propeller) of about 400nm thickness to be bound to F1 ATPase. Furthermore, coating of SU-8 with streptavidin and rotation assays tests with actin filaments are being carried out. The evolution of this micro device will permit the potential application of a new class of carrier with functionality in biological environment. Acknowledgement We thank all the contributors and sponsors for financial support. References [1] Hiroyuki Noji,Ryohel Yasuda, Masasuke Yoshida and Kazuhiko Kinoshita Jr. (1997) Nature 386: 299-302. [2] Ricky K Soong, George D Bachand, Hercules P Nerves et al (2000) Science 290: 1555-1558. [3] Brian H Cumpston, Sudaravel P Ananthavel, Stephen Barlow et al, (1999) Nature 398:51-54In:

Ni-NTA bead ATPase

SU-8 flagella

Fig. 1. Sketch of the ATP driven Micro device.

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68. Optical Control of Fluid Flow in Microfluidic Devices OROSZI 1, L., DÉR 1, A., RAKOVICS 2, V., VALKAI 1, S. AND ORMOS 1, P. 1 Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Temesvári krt 62, Szeged, 6726, Hungary, [email protected] 2 Institute for Technical Physics and Materials Science, Hungarian Academy of Sciences, Konkoly-Thege út 29-33, 1121 Budapest, Hungary

Electro-osmosis is an efficient means to move fluid in microfluidic channels. The flow is driven by the interaction of the electrical double layer at the channel wall with an electric field along the channel. The flow can be controlled by modifying the electrical parameters, either the charge of the channel wall or the electric field. If the surface chagre or the surface resistance of the channel wall is sensitive to light, the flow can be modulated by light. We have demonstrated this effect by using photoconductive surfaces. The resistance change due to the illumination changes the electric field above the photoconductive layer and consequently changing the rate of fluid flow. By using channels where upon a photoresistive CdS surface a linear PDMS channel was placed, flow rate changes of an order of magnitude were achieved. This gives serious possibilities for optical control of flow. We further developed the method by building channel structures of more complicated patterns, e.g. Y junctions. By appropriate illumination of the arms the flow direction could be selected between the arms optically, representing an optically controlled fluid switch. These elements: the linear channel and the switch can form the basis of more complex channel patterns with total dynamic optical control.. In a further development of the concept, by activating photoconductive layers of special pattern, the structure of the electric field and consequently the flow pattern could be manipulated by light. We created light activated helical flow patterns within a microfluidics channel. Such complex flow patterns offer the possibility of efficient mixing, a general problem in microfluidics due to the characteristically very low Reynolds number. We believe that the introduced concept of optically controlled electroosmosis opens up powerful possibilities in the area of microfluidics. Optical control offers the potential of building complex dynamically reconfigurable microfluidics systems. Reference Oroszi, L, Dér, A, Kirei, H, Rakovics, V, Ormos, P (2006) Appl. Phys. Lett. 89: 263508.

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69. Development of Flow Cytometric Multiplexed Microbead Assay for Detection of Mycrotoxin Contamination CZÉH 1, Á., TÖRÖK 1 L., GÖRÖMBEY 2 P., TÖRÖK 1 T., LANTOS 1 E., LUSTYIK 1,3 GY. 1 Soft Flow Hungary R&D Ltd., Pécs, [email protected] 2 Soft Flow Informatics, Debrecen, 3 University of Pécs, Faculty of Medicine, Department of Biophysics, Pécs,

A multiplexed, microsphere based, flow cytometric analytical assay has been developed for qualitative and quantitative detection of mycotoxin contamination (ochratoxin A, aflatoxin B1, fumonisin B1, T2-toxin and zearelenone) in food and feed products. Besides mycotoxins - the harmful products synthesized by various fungi species – the assay can be extended to determination of other contamining compounds such as antibiotics, hormones and genetically modified food components. Specific anti mycotoxin monoclonal antibodies were conjugated to 4.0 um, carboxyl-modified polystyrene particles (Duke Scientific) using three different conjugation techniques. A competitive ELISA type assay has been developed. The assay is based on the competition of the small mycotoxin antigen molecules and the mycotoxin-coupled PE (phycoerythrin) macromolecules. The quantitative model of the assay should take into account the significant difference of the lateral and rotational diffusion mobilities of the competing large and small molecules. In addition, it is also a significant difference compared to the similar competitive ELISA method that the number of antibody binding sites depends upon the number of polystyrene microparticles added to a given volume of assay sample. The multi-analyte assay uses multiplexed bead particle populations that are fluorescently labeled with gradually increasing concentration of a fluorescent dye, similar to the commercially available multiplexed assay kits. The measurements can be performed with any flow cytometry instrument that is capable of detecting PE fluorescence and the clustering fluorescence is around 670 nm. Our experiments were performed with FACSCalibur/CellQuestTM Pro and FACSArray Bioanalyzer instruments (BD Biosciences). New algorythms were developed and integrated into our FCAP Array software for clustering the acquired bead populations and processing the clusterized reporter fluorescence data.

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SINGLE MOLECULE TECHNIQUES, NANOTECHNOLOGY, MICROSCOPY AND IMAGING

70. Reactive Oxygen Species Generation in TiO2–Based Nanotube Suspensions under Different Physico-Chemical Conditions S. KURE 1, I. DOGŠA 1, P. UMEK 1, D. ARČON 1, F. BAJD 1, T. KONTE 1, D. STOPAR 2, J. ŠTRANCAR 1 1 „Jožef

Stefan“ Institute, Department for Condensed Matter, Jamova 39, SI-1000, [email protected] of Ljubljana, Biotechnical faculty, Department of Food Technology, Laboratory of Microbiology, Večna pot 111, SI-1000

2 University

Search for new effective disinfecting agents for surface coating is now in the focus of scientific research. The potential materials for this kind of application are based on TiO2. We synthesized TiO2-based nanotubes (TiNTs) by alkaline hydrothermal method. Under UV irradiation TiNTs show photocatalitic activity. TiNTs added to the suspension of E. coli (104 CFU/ml) doubled UV bactericidal effect by generating reactive oxygen species (ROS), as indicated by spin-trapping (DMPO) electron paramagnetic resonance (EPR). The magnitude of ROS generation was dependent on the crystalline form of TiO2, size of the surface area of the TiNTs, and on the wavelength of UV irradiation. The EPR signal increased up to 15-fold when wavelength was decreased from λ =365 nm to λ = 254 nm. This is in sharp contrast to the commercially available TiO2 suspension, where the EPR signal decreased 3-fold. Increasing the surface to mass ratio by ultrasonic treatment of TiNTs suspensions increased the dispersion stability for up to two days. Aggregate dissociation also increased EPR signal at higher pH values. At extreme pH values, however, the EPR signal was weak. This can be explained by the instability of DMPO-OH spin-trap adducts at extreme pH. On the other hand, the increased ionic strength increased EPR signal for only 10%, when concentration of monovalent ions at constant pH was doubled. Varying aggregation parameters of photocatalytic TiNTs is crucial factor in increasing antimicrobial surface coating properties.

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71. Cisplatin magnetosomes in combined chemotherapy and hyperthermia of tumour cells KALJAROVÁ, D., BABINCOVÁ, M. AND BABINEC, P. Department of Biophysics and Nuclear physics, Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynská dolina F1, Bratislava, 842 15, Slovakia, [email protected]

In this work session we suggest new method of combined chemotherapy and hyperthermia by cisplatin magnetosomes. Magnetic nanoparticles as vehicles for delivery of anticancer drugs after introduction to the tumour answer for increase of the efficacy and drug concentration at the tumour reduce the unpleasant site effects associated with chemotherapy [1]. We have prepared a surerparamagnetic colloid by milling magnetite particles and evaluated its heating capabilities in an alternating magnetic field with frequency 3,5 MHz. The mechanism of heating is based on Brownian relaxation (rotation of the particle as a whole according to external magnetic field) and Néel effect (orientation of the magnetic dipole moment thermally rotated inside the particles) [2]. As has been found relaxation losses are able to heat superparamagnetic suspension to 70 °C in about 100 minutes. The highest increase of temperature is observed at the ferrofluid with highest concentration of magnetite particles. Due to their superparamagnetic properties they are potential candidates as agents for electromagnetic hyperthermia (heating tissue to 41- 45 °C). Cisplatin is chemotherapeutic drug used to treat various types of cancers. Cisplatin acts by crosslinking DNA in various different ways making it impossible for rapidly dividing cells to duplicate their DNA for mitosis [3]. In Fig. 1 we compare efficiency of single therapies. With hyperthermia we observed 40 % and with chemotherapy 60 % death cells. The highest percentage of tumour cells decease was observed at combined therapy 98 %, where we used maximal concentration of cisplatin adsorbed on phosphate groups of definite quantity of magnetite nanoparticles. In our experiments we have demonstrated that combined therapy is from these the most suitable for treatment. Acknowledgement This work was supported by the research grant from the Slovak grant Vega No. 1/2012/05

Fig. 1. Dependence of tumours cells decease percent from various concentrations of magnetic particles, cisplatin and cisplatinmagnetosomes. The results are mean values from twelve independent measurements.

References [1] Babincová, M, Altanerová, V, Altaner, Č, Babinec, P: In vivo heating of magnetic nanoparticles in alternating magnetic field. Medical Physics, Vol. 31 (2004), 2219-2224 [2] Babincová, M, Šusteková, E, Babinec P, Čičmanec P, Craciun, V, Leszczynska, D: Heating of superparamagnetic colloid in high-frequency magnetic field: Implications for electromagnetic hyperthermia. Czechoslovak Journal of Physics, Vol. 50 (2000), 979-982 [3] Yan, X, Gemeinhart RA: Cisplatin delivery from poly (acrylic acid-co-methyl methacrylate) microparticles. Journal of Controlled Release, Vol. 106 (2005), 198-208

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72. Structure and Dynamics of Phospholipid Bilayers Incorporated into Layer-byLayer Polyelectrolyte Films. A Fourier Transform Infrared Spectroscopic study A. PILBAT 1, ZS. SZEGLETES 1, Z. KÓTA 1, V. BALL 2, P. SCHAAF 3, J-C. VOEGEL 2 AND B. SZALONTAI 1 Institute of Bophysics, Biological Research Centre, Hung. Acad. Sci., Temesvári krt. 26, Szeged, 6726, Hungary, [email protected] Université Louis Pasteur, Faculté de Chirurgie Dentaire, UMR 595, Institut National de la Santé et de la Recherche Médicale, 11, rue Humann, 67085 Strasbourg Cedex, France 3 Institut Charles Shadron, Unité Propre 22, Centre National de la Recherche Scientifique, 6 rue Boussingault, 67083 Strasbourg Cedex, France 1 2

Polyelectrolyte multilayers built layer-by-layer (LBL) adsorption offer a simple and versatile tool to have surfaces with adjustable properties (Decher 1232-37). It has been shown that by varying the electrolytes and/or the build-up conditions, the properties of the films, like thickness, cell adhesion, or protein adsorption (Schwinté et al. 11906-16) capacity can be altered at will. In attempts for practical applications, such films were functionalized with features ranging e.g. from anti-fungal activity, through anti-inflammatory properties to electro-optical devices. Considering the extensively charged nature of the polyelectrolytes in these films, there is no chance for direct incorporation of non-polar, hydrophobic compounds into them. From the point of view of practical applications, however, it could be very useful if such compounds, e.g. different proteins, peptides, drugs could be incorporated. For the incorporation of such protein molecules at least lipid bilayers are needed. If once such bilayers were formed in the interior of polyelectrolyte films, they might be utilized as controllable internal barriers as well. The present work describes the first experiments toward this goal. Dipalmitoyl phosphatidylcholine (DPPC) bilayer was created on the surface of an exponentially growing poly(glutamic acid)/poly(lysine) (PGA/PLL) layer-by-layer polyelectrolyte film (Gergely et al. 5575-82). The lipid bilayer decreased the surface roughness of the polyelectrolyte film. The layer-by-layer construction of the polyelectrolyte film could be continued on the top of the DPPC layer. We have shown that the lipid bilayer formed a barrier in the interior of the polyelectrolyte film, which blocked the diffusion (a prerequisite for exponential growth) of the polyelectrolytes. Thus, a new growth regime started in the upper part of the polyelectrolyte film, which was added to embed the DPPC bilayer. The structure and the dynamics of the DPPC bilayer on the polyelectrolyte film surface remained similar to that of its hydrated multi-bilayers, except that the phase transition became wider. In the case of embedded DPPC bilayers, in addition, the phase transition temperature also decreased. This is the result of interactions with the non-concerted movements of the barrier-separated lower and higher parts of the polyelectrolyte film. Gramicidin A (GRA) as a model of lipid-soluble peptides and proteins was successfully incorporated into such DPPC films. The DPPC films, either with or without GRA, were remarkably stable, as many heating-cooling cycle to measure phase transition could be carried out without visible alterations as wanted. Acknowledgement This work was carried out within the frame of the bilateral Franco-Hungarian project “Balaton” (F-34/2005). References Decher, G. "Fuzzy nanoassemblies: Toward layered polymeric multicomposites." Science 277.5330 (1997): 1232-37. Gergely, C., et al. "Human serum albumin self-assembly on weak polyelectrolyte multilayer films structurally modified by pH changes." Langmuir 20.13 (2004): 5575-82. Schwinté, P., et al. "Stabilizing effects of various polyelectrolyte multilayer films on the structure of adsorbed/embedded fibrinogen molecules: An ATR-FTIR study." Journal of Physical Chemistry B 105.47 (2001): 11906-16.

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73. High resolution imaging and elemental analysis of PAGE electrophoretograms by scanning proton microprobe KOCSONYA 1, A., KOVÁCS 1, I., SZİKEFALVI-NAGY 1,Z., HOPFF 2, D., LÜTHJE 2, S., AND NIECKE 3, M. KFKI Research Institute for Particle and Nuclear Physics, Budapest, Hungary, [email protected] Universität Hamburg, Biozentrum Klein Flottbek und Botanischer Garten, Hamburg, Germany 3 Universität Hamburg Institut für Experimentalphysik, Hamburg, Germany 1 2

The PIXE-PAGE technique developed for elemental analysis for protein bands in gel electrophoretograms was adapted for scanning proton microprobe. In the electrophoretogram the proteins and protein fragments are separated in narrow bands and the metal ions bound to the proteins can be detected and in some cases quantified by PIXE spectroscopy [1]. The PIXE-PAGE method adapted for scanning proton microprobe provides two-dimensional mapping of the trace metals in the protein bands. Further advantage of the microPIXE-PAGE is that the fast continuous scanning reduces the thermal deterioration of the sample and the artefacts due to dust-like impurities can be filtered out in the data evaluation process. For the PAGE samples a large area (80 mm2) Si(Li) detector of 220 mSr was installed behind the gel sample in the target chamber of the Hamburg-Budapest microprobe. An optimized combination of C and Be foils was applied as beam stopper between the thin gel sample and the detector to diminish background due proton stopping. The microPIXE-PAGE method was tested on myoglobin and cytochrome c electrophoretograms. A series of test measurements were performed to optimize the beam current and to determine the Fig. 1. Fe map of cytochrome c band detection limit. For the test measurements the total amount of the protein loaded on the top of the polyacrylamide gel were varied from 3 to 30 µg. The two dimensional Fe map of the band of the 10µg cytochrome c is shown in Fig. 1. The total Fe content of the band determined by PIXE analysis was in good agreement with the amount of the protein loaded on the gel. With optimized parameters we achieved a detection limit of 0.9 ng for Fe in PAGE. Acknowledgement Special thanks are expressed to Dr. Cs. Bagyinka for the myoglobin sample. This work was partially supported by the Hungarian National Research Fund (OTKA) under Research Contract No. T037825, and the IAEA under Research Contract No.13260, and a PhD student’s grant from the University of Hamburg (HmbNFG) to D.H. References [1] Szıkefalvi-Nagy, Z, Demeter, I, Bagyinka, Cs, and Kovács, K.L, (1987) Nucl. Instr. and Meth.. B 22: 156-158

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74. Fenestration of endothelial cells detected with AFM BODOR 1, C., NAGY 2, J., MASSZI 1, A., JENEI 2, A, MIRZAHOSSEINI 1, S., MÁTYUS 2, L., AND ROSIVALL 1, L. Institute of Pathophysiology, Hungarian Academy of Sciences and Semmelweis University Pediatric and Nephrology Research Group, Budapest, Hungary. 2 Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, P.O. Box 39, 4012 Debrecen, Hungary. [email protected] 1

Atomic Force Microscope was used to investigate the topology of cell surface of endothelial cells. The juxtaglomerular (JG) portion of afferent arteriole (AA) facing the renin expressing granular cells is fenestrated. These pores are thought to play a pivotal role in the function of JG apparatus. To elucidate the intracellular mechanisms responsible for endothelial fenestration human umbilical vein endothelial cells were isolated and cultured and treated with recombinant VEGF. The transendothelial permeability was measured by using fluorescently labelled dextran. To detect fenestration, cells were treated with VEGF or its solvent. After fixing with ethanol, cell surface was screened by atomic force microscopy (AFM). Activation of p38 kinase was detected by applying phospho-specific antibody and Western blotting. Endothelial permeability increased by more than 2-fold subsequently to VEGF treatment. This effect was detectable as early as after 15 minutes. In control cultures, only a few fenestrae could be visualized by AFM. However, after 15 min of VEGF treatment we detected increased fenestration which further increased after 48 hours of VEGF treatment. The total area of fenestrae also increased in VEGF treated cultures. The average size of these pores was 120-160 nm. VEGF induced rapid and sustained activation of p38 detectable even after 48 hours. Preincubation with SB203580, an inhibitor of p38 reduced VEGF-induced permeability measured by dextran diffusion. Moreover SB203580 significantly inhibited formation of fenestrae of VEGF treated cells. In our experiments AFM is proved to be a useful technique for the characterization of endothelial fenestration, to gain high-resolution images of the fenestrae. The results suggest that p38 plays an important role in the regulation of VEGF induced fenestration.

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75. Interaction between charged membrane surfaces mediated by charged rod likenanoparticles J. PAVLIČ 1,2, A. IGLIČ 1, V. KRALJ-IGLIČ 3,K. BOHINC 1,2 Faculty of Electrical Engineering, Tržaška 25, 1000 Ljubljana, Slovenia, [email protected] University College for Health Studies, Poljanska 26a, 1000 Ljubljana, Slovenia 3 Faculty of Medicine, Lipičeva 2, Ljubljana, Slovenia 1

2

The force between charged membrane surfaces, separated by a solution of charged rod-like nanoparticles was studied. The rod-like nanoparticles were assumed to have spatially distributed electric charge. The nonlocal Poisson Boltzmann (PB) theory for the rod-like nanoparticles was developed where the finite size of the rod-like nanoparticles was taken into account. It was shown that for large enough membrane surface charge densities and large enough dimensions of rod-like nanoparticles, the force between equally charged membranes may be attractive due to the spatially distributed charges within the nanoparticles. Some experiments were conducted for comparison with theoretical conclusions.

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THEORETICAL BIOPHYSICS, BIOINFORMATICS AND COMPUTER MODELLING

TEORETICAL BIOPHYSICS, BIOINFORMATICS & COMPUTER MODELLING

76. A binary integer programming relaxation for the weighted maximum compatibility problem BUSA-FEKETE 1, R., KOCSOR 1, A. AND BAGYINKA 2, CS. Research Group on Artificial Intelligence of the Hungarian Academy of Sciences and University of Szeged, Aradi vértanúk tere 1., Szeged, 6720, Hungary, [email protected] 2 Institute of Biophysics, Biological Research Center, Temesvári krt. 62., Szeged , H-6726, Hungary 1

In evolutionary studies it is a commonly used technique to represent a collection of phylogenetic trees with a single tree. These algorithms are the so-called consensus tree methods. The first consensus tree method was proposed by Adams in 1972 [1], and since then many methodologies were introduced that aim to give one ‘representative’ tree for a given set of phylogenetic trees [2]. The most widespread methods are the strict consensus and majority consensus which are also included in Phylip [3] and Paup [4]. Each edge of a phylogenetic tree over a set of object X corresponds to a bipartition on it. So the consensus methods work on a set of bipartitions or in other words on a set of binary (two-state) characters C which are obtained by the phylogenetic trees under question. If the binary characters are compatible ( A pair of binary full character or bipartition A | B ⊆ X and C | D ⊆ X are compatible if at least one of the A ∩ C, A ∩ D, B ∩ C and B ∩ D is the empty set.) then we can represent a binary character set C exactly using a single (not necessarily binary) tree. Therefore one tries to find the largest number of compatible binary characters. This problem is NP-hard [5], and it is known as Largest Compatible Subset Problem (LCSP). In this study we deal with a related instance of the LCSP: in addition to the original problem, we also have an associated non-negative real-value weight function w : C → ¡ + on the set of binary characters. We seek the C’ compatible subset of C that maximizes the sum of the weights of characters containing C’. In the simplest case the weight function can mean the number of trees they contain from the input trees, or it can also give a value to a character according to the likelihood score of the input trees. We give a binary integer programming relaxation of this problem, and solve it applying the well-known Branch and Bound algorithm [6] which is a general method for finding the optimal solution of different combinatorial optimization problems. We tested our methodology using the Quartet Puzzling [7] and a parsimony method from the PAUP Program Package. Both of these methods produce more than one output trees for the input dataset. We compared the consensus tree methods on the output of them using various sizes of trees and various evolutionary models [8, 9]. We also investigated the performance of our consensus method when we use different weighting functions (e.g. parsimony-based or likelihood-based). The experiments clearly showed that the consensus tree algorithm introduced here improves the performance of the tree-building methods, has moderate time consumption (proportional to the tree building method itself), and thus it is suitable for a post-processing step of a phylogenetic analysis tool. References [1] Adams, EN, (1972) Systematic Zoology, 21:390-397. [2] Bryant, D (2003) BioConsensus, DIMACS. AMS. 163-184. [3] Phylip program package, http://evolution.genetics.washington.edu/phylip.html [4] Paup program package, http://paup.csit.fsu.edu/ [5] Day, W. and Sankoff, D. (1986): Systematic Zoology, 35(2):224-229 [6] Land, AH. and Doig AG. (1960): Econometria, 28:497-520 [7] Schmidt, H.A., Strimmer K., Vingron M., and von Haeseler A. (2002): Bioinformatics. 18:502-504. [8] Hasegawa, Kishino & Yano (1985): J Mol Evol., 22(2):160-74. [9] Felsenstein (1981): J. Mol. Evol., 17(6):368-76.

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77. Binding similarities between calmodulin antagonists and calmodulin target complexes revealed by molecular dynamics simulations ISTVÁN KÖVESI, MONIQUE LABERGE, JUDIT FIDY Department of Biophysics and Radiation Biology, Faculty of Medicine, Semmelweis University, Puskin u 9, Budapest, 1088, Hungary,

[email protected]

In our previous study we reported 10 ns molecular dynamics simulations of two kinds of calmodulin (CaM) antagonists bound to CaM, namely trifuoperazine (TFP) and N-(3,3,diphenylpropyl)-N’-[1-R-(3,4bis-butoxyphenyl)-ethyl]-propylenediamine (DPD). Our results in that study showed, which antagonist binds more efficient and what are the agents, which determine, whether TFP or DPD is more efficient. In our recent study we report the similarity of binding an antagonist and the target binding, and which CaM – antagonist complex is similar to the CaM – target complex. To answer this question all-atom simulations were carried out with physiological salt concentration at constant pressure and temperature. During the simulation CaM-target complex also adopts a stable conformation. The conformation of CaM – Target complex is more similar to CaM – 2DPD complex than CaM – 2TFP. The binding is also more hydrophobic in the case of CaM-target, so the nonbond interactions are also more similar to CaM-2DPD complex, which agrees with our previous results, where the 2 CaM antagonist complexes were compared. These results show that the role of hydrophobic interactions are essential for the antagonist or target binding of CaM, which suggests a drug design startegy for developing new anti-CaM molecules.

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78. Theoretical study of coumarine β-cyclodextrin inclusion complexes HOLUBEKOVA 1, A., MACH 1, P. AND URBAN 1, J. 1

Faculty of Mathematics, Physics and Informatics, Comenius University, Mlynska dolina, 84248 Bratislava, Slovakia, [email protected]

Cyclodextrins (CDs) are cyclic oligosaccharides composed of several d-glucose units bonded by α(1,4) linkages. The most common natural CDs are α-, β- and γ-CDs consisting of six, seven and eight Dglucopyranose residues, respectively. From a topological point of view, Beta-cyclodextrin (β-CD) can be described as a truncated cone, in which the narrow rim (6.4 Å) bears the primary hydroxyl group whereas the wide rim (15.4 Å) bears the secondary OH groups. Since no hydroxyl group is present within the toroidal cavity of β-CD, this zone of the molecule has a pronounced hydrophobic character. The unique shape and physical–chemical properties of the cavity, together with van der Waals forces and hydrogen bonding allows the formation of inclusion complexes with a wide range of compounds, where the extent of the complex formation depends on the polarity of the absorbed molecules. The derivatives of coumarin (coumarins) usually occur as secondary metabolites present in seeds, root, and leaves of many plant species, although their presence has also been detected in microorganisms and animal sources. Because of the structural diversity found in this family of compounds they are used in many areas, as additives to food and cosmetics, optical brightening agents, dispersed fluorescent, laser dyes and for medical purposes as blood thinner to keep blood flowing smoothly and prevent the formation of blood clots, as anti-fungicidal and anti-tumors. Coumarins are very convenient also for the study of molecular complexes because of their spectroscopic activities. If coumarin enters the cavity of β-CD its spectra changes due to bonding with β-CD. Our contribution presents quantum chemical calculations of the structures of inclusion complexes of βCD with coumarins, (coumarin-6, coumarin-30, coumarin-47, and coumarin-522). On the basis of semiempirical PM3 method and DFT theory the optimal geometries have been found. Molecular dynamics study has been applied in the case of the β-CD coumarin-522 inclusion complex.

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79. Interdomain interactions influence the subunit dynamics of PGK BALOG 1, E., PALMAI 1, Z. AND FIDY 1, J. Department of Biophysics and Radiation Biology, Faculty of Medicine, Semmelweis University, Puskin u 9, Budapest, 1088, Hungary,

[email protected]

Molecular dynamics simulation was performed to examine the inter- and intradomain motions of the twodomain enzyme yeast phosphoglycerate kinase without the presence of substrates. To elucidate contributions from individual domains, simulations were carried out on the complete enzyme as well as on each isolated domain. The enzyme is known to undergo a hinge-bending type of motion as it cycles from an open to a closed conformation to allow the phosphoryl transfer occur. Analysis of the correlation of atomic movements during the simulations confirms hinge bending in the nanosecond timescale: the two domains of the complete enzyme exhibit rigid body motions anticorrelated with respect to each other. The correlation of the intradomain motions of both domains converges, yielding a distinct correlation map in the enzyme. In the isolated domain simulations—in which interdomain interactions cannot occur—the correlation of domain motions no longer converges and shows a very small correlation during the same simulation time. This result points to the importance of interdomain contacts in the overall dynamics of the protein. Acknowledgement Financial support from the Hungarian Academy of Sciences (one-year fellowship, B.E.), and from the Hungarian grant No. 512/2006 of the Scientific Committee of the Ministry of Health, Hungary, (F.J.) is highly appreciated.

Fig. 1. X-ray structure if yPGK

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80. Effect of ligand binding on the compressibility of dihydrofolate reductase studied by molecular dynamics simulation BALOG 1, E., FIDY 1, J. AND PERAHIA 2, D. 1

Department of Biophysics and Radiation Biology, Faculty of Medicine, Semmelweis University, Puskin u 9, Budapest, 1088, Hungary,

2

Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université Paris-Sud, Bât 430, Orsay, 91405, France

[email protected]

The change in compressibility of dihydrofolate reductase (DHFR) on binding the ligand methotrexate (MTX) is determined by molecular dynamics simulations. DHFR has been recognized as a drug target for inhibiting DNA synthesis in rapidly proliferating cells such as cancer cells, while MTX has been used effectively as a cytotoxic agent in the treatment of cancers. Based on MD simulations the compressibility of the complex is significantly higher compared to the unbound protein, which is in agreement with previous experimental results. Our data reveal that the softening of the complex upon ligand binding is mainly caused by the internal cavity formations in the active site of the protein. This suggests a different picture of the ligand binding to proteins than the generally accepted tight “key-lock” model. Acknowledgement Financial support from HAS – CNRS collaborative grant is highly appreciated.

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81. Secondary Structural Motifs of Polyalanine and Polyglutamine Peptides LEITGEB 1, B., KERÉNYI 1, Á., BOGÁR 2, F., PARAGI 2, G., PENKE 2,3, B. AND RÁKHELY 1,4, G. Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62, Szeged, H-6726, Hungary, [email protected] 2 Supramolecular and Nanostructured Materials Research Group of the Hungarian Academy of Sciences, University of Szeged, Dóm tér 8, Szeged, H-6720, Hungary 3 Department of Medical Chemistry, University of Szeged, Dóm tér 8, Szeged, H-6720, Hungary 4 Department of Biotechnology, University of Szeged, Temesvári krt. 62, Szeged, H-6726, Hungary 1

Polyalanine and polyglutamine peptides are ranked among the homopolymeric amino acids (HPAAs), which are composed of the same amino acids, namely alanine (Ala) and glutamine (Gln) residues, respectively. These two HPAAs are well-known to cause a variety of human illnesses and to play a relevant role in the formation of several neurodegenerative diseases [1,2,3]. Therefore, the aim of this study was to investigate the structural motifs and intramolecular H-bonding patterns of these HPAAs. The conformational study of poly-(Ala) and poly-(Gln) peptides consisting of 7, 10, 14 and 20 residues, respectively, was carried out by means of simulated annealing (SA) calculations. The HPAAs were modelled in two different forms: either with charged N-terminal amino and C-terminal carboxyl groups, or with the Nand C-terminal ends blocked by acetyl and N-methyl amide groups, respectively. For the poly-(Ala) and poly-(Gln) peptides, few secondary structural elements (including type I and type III β-turns, α-helix, 310-helix and antiparallel β-strand) were identified. β-turns could be found along the entire sequence of HPAAs, furthermore, several conformers containing two or more β-turns, either consecutively or separately, were observed. In the case of helices and strand, segments of different lengths characterized by these periodic secondary structures were determined. Beside the conventional secondary structural elements, other motifs occurred in the tripeptide units were examined. In accordance with the presence of β-turns, 310- and α-helical segments, characteristic H-bonding patterns could be found for the conformers of poly-(Ala) and poly-(Gln) peptides. In both HPAAs, mainly i←i+3 and i←i+4 H-bonds evolved between the backbone CO and NH groups were observed. Further intramolecular H-bonds were identified for the poly-(Gln) peptides, which were formed by the participation of CO and NH groups of Gln side-chains. The results of our conformational study pointed out that the poly-(Ala) and poly-(Gln) peptides could be characterized by several secondary structural elements, and the intramolecular H-bonding patterns were in agreement with the presence of different secondary structures for both HPAAs. Acknowledgement This research was supported by grants GVOP-3.1.1.-2004-05-0492/3.0 and RET 08/2004. References [1] Brown, LY, Brown, SA (2004) Trends Genet 20: 51-58. [2] Perutz, MF (1996) Curr Opin Struct Biol 6: 848-858. [3] Perutz, MF (1999) Trends Biochem Sci 24: 58-63.

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82. Comparative Conformational Study of the Eight Stereoisomers of the Antimicrobial Peptide, Indolicidin KERÉNYI 1, Á., RÁKHELY 1,2, G. AND LEITGEB 1, B. Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62, Szeged, H-6726, Hungary, [email protected] 2 Department of Biotechnology, University of Szeged, Temesvári krt. 62, Szeged, H-6726, Hungary 1

Indolicidin (H-ILPWKWPWWPWRR-NH2) is an antimicrobial tridecapeptide isolated from bovine neutrophils [1]. This peptide possesses a broad spectrum of antimicrobial activity against bacteria and fungi, nevertheless, it exhibits haemolytic and antiviral activities. Data derived from the previous experimental and theoretical studies led to considerably diverse conclusions regarding the secondary structure of indolicidin [2,3,4]. Therefore, our aims were to investigate the secondary structural elements of this antimicrobial peptide and to examine the effect of cis-trans isomerization on the conformational properties of indolicidin. Simulated annealing (SA) calculations were performed, in order to study the structural features of indolicidin possessing trans or cis Xaa-Pro peptide bonds. In accordance with the cis-trans isomerization of three Xaa-Pro peptide bonds located in this antimicrobial peptide, eight different isomers could be distinguished, therefore the SA simulations were carried out on each isomer, respectively. In the case of trans isomers, tpye I and type III β-turns were found in the tetrapeptide units possessing Pro in the second position. Furthermore, 310- and poly-proline II helical segments were also detected along the sequence of peptides having trans Xaa-Pro peptide bonds. For the cis isomers, type VI β-turns were observed in tetrapeptide units containing Pro in the third position. Additionally, in the case of both trans and cis isomers, β-turns and 310-helical segments appeared in tetrapeptide units possessing Pro in the first position, especially at the C-terminal part of peptides. According to the occurrences of tpye I and type III βturns as well as 310-helical segments, i←i+3 H-bonds evolved between the backbone atoms were identified in the above-mentioned tetrapeptide units for both isomers. Proline-aromatic interactions could be formed between the pyrrolidine ring of Pro amino acid and the aromatic ring of preceding or following Trp residue. In the case of Pro-Trp sequence, a number of such interactions was found in both trans and cis isomers, while for the Trp-Pro units, large populations of these interactions were observed only for cis isomers. The latter proline-aromatic interactions contribute to the stability of type VI β-turns. The comparative conformational analysis of trans and cis isomers of indolicidin revealed that different secondary structures and stabilizing intramolecular interactions could be observed for both isomers. However, the C-terminal part of trans and cis forms seemed to show similar structural properties. References [1] Selsted, ME, Novotny, MJ, Morris, WL, Tang, Y-Q, Smith, W, Cullor, JS (1992) J Biol Chem 267: 4292-4295. [2] Falla, TJ, Karunaratne, DN, Hancock, REW (1996) J Biol Chem 271: 19298-19303. [3] Bahng, MK, Cho, NJ, Park, JS, Kim, K (1998) Langmuir 14: 463-470. [4] Ladokhin, AS, Selsted, ME, White, SH (1999) Biochemistry 38: 12313-12319.

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THEORETICAL BIOPHYSICS, BIOINFORMATICS AND COMPUTER MODELLING

83. Investigation of the Structural and Conformational Features of Arg- and Lysconopressins LEITGEB 1, B., JANCSÓ 1, G., KERÉNYI 1, Á., AND RÁKHELY 1,2, G. Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62, Szeged, H-6726, Hungary, [email protected] 2 Department of Biotechnology, University of Szeged, Temesvári krt. 62, Szeged, H-6726, Hungary 1

Arg-conopressin-S (H-CIIRNCPRG-NH2) and Lys-conopressin-G (CFIRNCPKG-NH2) are cyclic disulfide-bridged nonapeptides, which were isolated from the venom of fish-hunting cone snails, Conus striatus and Conus geographus, respectively [1]. Although, their biological effects have been examined in detail, data derived either from experimental or from theoretical investigations have not been published so far, concerning the three dimensional structure of these conopeptides. Thus, our aims were to perform a detailed conformational analysis on the conopressins and to identify their structural and conformational features. To explore the conformational spaces of two conopeptides, simulated annealing (SA) and molecular dynamics (MD) calculations were carried out. As both peptides contain Pro amino acid in seventh position, therefore the cis and trans isomers of conopressins were modelled, respectively, according to the cis-trans isomerization of Cys6-Pro7 peptide bond. In order to characterize the Φ-Ψ conformational spaces and the distributions of conformers, Ramachandran and three dimensional Ramachandran plots were constructed using the Φ and Ψ torsion angles of amino acids. Additionally, applying these plots and the conformational similarity indices, the conformational distributions were compared to each other. For the side-chains of Cys1, Ile2/Phe2, Ile3, Arg4, Asn5, Cys6 and Lys8/Arg8 amino acids, the ratios of g(+), g(-) and trans rotamer populations were determined. In the conformers of conopressins, different types of β-turns located in the certain tetrapeptide units were identified, and several types of them were found to be stabilized by characteristic intramolecular H-bonds. However, further H-bonds were also detected, which could contribute to the stabilization of the structure of these conopeptides. For the conformers, cluster analysis was performed, in order to determine the conformationally related subfamilies and to identify their representative structures. Based on the MD trajectories, the dynamic behaviour of the structural and conformational properties of conopressins were examined. In our study, which is the first structural investigation of Arg- and Lys-conopressins, we provide a detailed characterization of the various structural and conformational features of conopressins, which might lead to better understanding of the bioactivity of these conopeptides at the molecular level. References [1] Cruz, LJ, de Santos, V, Zafaralla, GC, Ramilo, CA, Zeikus, R, Gray, WR, Olivera, BM (1987) J Biol Chem 262: 15821-15824.

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153

POSTERS

THEORETICAL BIOPHYSICS, BIOINFORMATICS AND COMPUTER MODELLING

84. Effect of Catheter Orientation on Reconstruction Accuracy and Dosimetry in CT-based Breast Brachytherapy FRÖHLICH 1,2, G., MAJOR 2, T., POLGÁR 2, CS. AND FODOR 2, J. 1 2

Semmelweis University, Doctorial School, Üllıi út 26., Budapest, 1085., Hungary, [email protected] National Institute of Oncology, Department of Radiotherapy, Ráth György utca 7-9., Budapest, 1122., Hungary

Introduction: To investigate the effect of catheter orientation related to CT scanning plane on catheter reconstruction and dosimetry in interstitial high-dose-rate breast brachytherapy. Materials and methods: A: Five catheters arranged in triangular configuration were CT scanned with 3 mm slice thickness in different positions related to the plane of CT scanning. The catheters perpendicular to CT slices was selected as reference position. Catheters were reconstructed and different dose plans with no optimization (Paris dosimetry system, PDS), geometrical (GOS) and dose point optimization (DPO) were created. Treatment time (ttreat) and volume irradiated by the reference dose (Vref) were calculated and compared. B: After that, ten catheters implanted in a special phantom simulating a target volume were used. The phantom setup was rotated in different angles on the CT couch. First, the catheters were parallel to CT scanning plane, and then they were angled in 1-2, 20, 40, 60 and 90 degrees. 3 and 5 mm CT slice thickness were used. The catheters were reconstructed and the target volume was outlined in each slice. Using three optimization methods (PDS, GOS, DPO) treatment plans were created, dose-volume parameters (Vref, V1.5xref, V1.5xMCD, VPTV, V100, V150, D90 and Dmin) and doses in marker points (D1, D2, D3) were calculated and compared. Results: A: The largest deviation in ttreat and Vref were observed when the catheters were rotated by only 1-2 degrees from the parallel position. For ttreat 2.9%, 2.5% and 1.3% and for Vref 4.3%, 3% and 2.3% was found at PDS, GOS and DPO method, respectively. At all other orientations the deviation was less than 2% for ttreat and 3% for Vref. B: The volumes of PTV were in agreement within 1% at 3 mm regarding all orientations, and the maximum deviation to mean was less than 1.5% at 5mm. Mean volume at 3 mm slice thickness was close to the real volume and at 5 mm thickness it was around 5% less. No significant differences were found between dose-volume parameters obtained at 3 and 5 mm slice thickness. In the marker points the maximum deviation is only 4 % in GOS, but we found no relation between the catheter orientation and the point doses. In the dependence of the dose-volume parameters on the angle in GOS no trend was found and this optimization resulted in the smallest deviations for all parameters, the maximum deviation was 5 %. Conclusions: The best catheter orientation regarding geometrical and dosimetrical accuracy would be the perpendicular or parallel catheter positions, which is rarely realised in clinical practice. More accurate catheter reconstruction can be achieved with larger angle between the catheters and CT scanning plane. The CT based catheter reconstruction results in acceptable accuracy if the slice thickness is 3 mm. The effect of catheter orientation on dose parameters in geometrically optimized system is less than in the Paris and dose point optimized systems.

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154

POSTERS

THEORETICAL BIOPHYSICS, BIOINFORMATICS AND COMPUTER MODELLING

85. The Twilight Zone Between Order and Disorder SZILÁGYI, A. AND GYÖRFFY, D. Institute of Enzymology, Hungarian Academy of Sciences, Karolina út 29, Budapest, H-1113 HUNGARY, [email protected]

Intrinsically disordered proteins and protein segments often have a markedly different amino acid composition from that of ordered proteins. This observation is the basis of several disorder prediction methods. However, these predictions often fail, especially for shorter sequences. In this work, our goal is to investigate the relationship between amino acid composition and order/disorder in more detail. For a more theoretical approach, we analyzed the order/disorder behavior of model proteins. Two types of two-dimensional lattice models were used: the conventional hydrophobic-polar (HP) model and a model employing three monomer types: hydrophobic, positively charged and negatively charged (we call this the HPN model). We generated all possible sequences of lengths 4 to 24, and used exact enumeration to find the ground state energy of each sequence. For longer chains, the ground state energies were approximated and sequence space was sampled. Sequences having a ground state energy per monomer higher than a predefined threshold were considered as intrinsically disordered. Looking at the relationship between amino acid composition and disorder, we found a chain length dependent transition from disorder to order in both models. For chains with a given length, there is a well-defined region in amino acid composition space where most sequences with the given amino acid composition are ordered, and another region where most are disordered. The region between these two regions is a "twilight zone": sequences with amino acid compositions in the twilight zone can be ordered or disordered, depending on the specific sequence. Both the width and the position of the twilight zone depend on chain length: shorter proteins have a twilight zone wider than that of longer proteins, and shifted towards higher hydrophobicity and lower charge. A closer look at the conformations of the ground states of short and long chains shows that this finding is the result of different organizing principles. Long chains can easily form a large hydrophobic core that can incorporate additional H monomers almost independently from their positions in the sequence. New charges are also more easily accomodated. Short chains, however, have smaller, if any, hydrophobic cores, and are more dependent on specific interactions and sequence patterns for stability. Turning to real proteins, we examine the distribution of points corresponding to ordered and disordered proteins of various lengths in both amino acid composition space and on the charge-hydrophobicity plot. The findings are in accordance with the theoretical predictions: a twilight zone can be observed, and its width decreases with growing chain length. The results point to the role of protein size in determining order/disorder in proteins, as illustrated by the example of two-state dimers (dimeric proteins whose chains are disordered in monomeric form but fold upon association). In addition, the results demonstrate the inherent limitations of amino acid composition based disorder prediction methods. To improve predictions, chain length or protein size should be taken into account. For small proteins, predictions could be improved by predicting specific interactions between residues.

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155

POSTERS

THEORETICAL BIOPHYSICS, BIOINFORMATICS AND COMPUTER MODELLING

86. Deconvolution of fluorescence spectra as a tool in structural investigations of compounds of different complexity RADOTIĆ 1, K., DJIKANOVIĆ 1, D., KALAUZI 1, A. AND JEREMIĆ 2, M. 1Centre 2Faculty

for Multidisciplinary Studies, University of Belgrade, Despota Stefana 142, 11000 Beograd, Serbia, [email protected] of Physical Chemistry, University of Belgrade, 11000 Beograd, Serbia

Fluorescence spectroscopy is a sensitive tool for structural and kinetic studies of both small molecules and macromolecules. The analysis of fluorescent spectra of polymeric molecules is especially complex, since they may contain different fluorophores or fluorophores in various microenvironments. Therefore, it is of crucial importance to use a valid mathematical model of the emission spectrum of one fluorophore, and then to apply such model in deconvolution of the complex spectra. Some of the widely used analytical expressions describing the components, such as Gaussian and Lorentzian, suffer from a basic drawback, since they are symmetric, contrary to the emission spectra, which are asymmetric. In this work we compared various models for deconvolution of fluorescence spectra of a simple fluorophore. On the basis of our previous results on the utilisation of symmetric model in analysis of the complex emission band shapes (Radotić et al 2006), we introduced the new model obtained by combination of symmetric and asymmetric models. The model is tested on molecules of different complexity, in order to confirm its reliability for complex spectra. We analysed fluorescence spectra of simple molecules containing one fluorophore: coniferyl alcohol and tryptophane, molecules containing two fluorophores: ferulic acid (a constituent of natural lignin), as well as complex multifluorophore molecules: dehydrogenative polymer (DHP) obtained by polymerisation of coniferyl alcohol and isolated lignin. All investigated species belong to benzene-substituted class of compounds, and it is reasonable to assume that they have similar fluorescence band contour. Technically, for each molecular species studied, a series of emission spectra were measured by varying excitation wavelengths with 5 nm steps, starting from 360 nm. In this way, fluorophores in the molecule are gradually excited. Results presented indicate that even in case of simple emission spectra one cannot rely on the positions of symmetric components for tentative fluorophore identification. In complex cases, such as multifluorophore polymers, they may be used only as an indicator of the discrete nature of emission, or eventually assessment of the number of fluorophores, only when corroborated by other experimental approaches (Radotić et al 2006). Using correct asymmetric models may mean a step forward in identifying the existing fluorophores in complex molecules, which may be a valuable structural tool. Acknowledgement. Grant 143043 of the Ministry of Science and Environmental protection of the Republic of Serbia supported this study References [1] Radotić, K., Kalauzi, A., Djikanović, D., Jeremić, M., Leblanc R., Cerović, Z. (2006) J. Photochem. Photobiol. B: Biology 83: 1-10

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156

POSTERS

THEORETICAL BIOPHYSICS, BIOINFORMATICS AND COMPUTER MODELLING

87. Analysis of the Centre of Pressure Movements of Standing Subjects SEVSEK, F., AND RUGELJ, D. University of Ljubljana, University College of Health Studies, Poljanska 26a, Ljubljana, Slovenia, [email protected]

Measurement of the human body centre of pressure (COP) movement with a force platform (stabilometry) is a standard procedure for the assessment of postural stability. Here a subject stands still on a special platform that is mounted on pressure sensors transmitting data via analogue to digital converter to a computer where the time dependence of the COP trajectory (postural sway) is recorded. Anyhow, it is still an open question how to extract physiological relevant information from the experimental data. Recently we proposed a method where the outline of the sway region is expressed in terms of Fourier coefficients [1] that are determined by asymmetric fitting considering minimal outline bending energy [2,3]. In this study it is shown that the postural sway data can be reproduced by considering random movement of the COP within a central ellipsoidal region whereas outside it the positions are accepted only with the probability exp(-E/T). E represents the energy and is proportional to the square of the distance from the central ellipsoidal region whereas T plays the role of temperature. Simulated data were compared to the measurements of 41 elderly (60 to 80 years) and 20 young (20 to 25 years) subjects where the influence of different sensory inputs was studied by performing the experiments with the subjects standing on a hard and compliant surface with their eyes open and closed. From the COP trajectories the outlines and areas were calculated and compared to the sway areas as calculated by the principal component analysis [4]. For all the experimental conditions this area ratio, which is related to the shape of the sway area, was for 60 s measurements typically close to 2 for young subjects and about twice as large, with much larger standard deviation, for elderly subjects. The exact value of this ratio was used to determine the temperature parameter (T) from the simulated data. Acknowledgement The research was supported by the Slovenian Research Agency (contract No. J3-6423-0382-06). References [1] Sevsek F , Gomiscek G (2004) Comput. methods programs biomed. 3: 189-194. [2] Rugelj D, Sevsek F (2007) WSEAS transactions on signal processing 3: 213-219. [3] Sevsek F (2007) WSEAS transactions on information science and applications 4: 794-799. [4] Oliveira L et al. (1996) Physiol. Meas. 17: 305-312

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POSTERS

MISCELLANY

MISCELLANY

88. Measurement of visceral fat thickness by ultrasound and comparison with visceral fat area determinations SZEBENI ÁGNES, HALMY L. IRM Central Hospital, Budakeszi Str 48/B., Budapest, H-1121. Hungary, [email protected]

Introduction: Visceral fat quantity is an important factor in the prediction of the cardiovascular risk of obesity. The „gold standard” of its measurement is the determination by CT. However, the exposition to ionizing radiation, high cost and low availability put limits to the routine use of CT for this purpose. Ultrasonography (US) is a simple, readily available, low-cost method for the measurement of visceral fat thickness (VFT) which correlates well with the CT data, according to the literature. Another simple and available method is the bioimpedance measurement of visceral fat area (VFA). Purpose: Comparison of VFT and VFA measurements and further, study of correlations betveen VFT and US attenuation of the liver, subcutaneous fat thickness (SCF), body mass index (BMI) and serum lipid data. Material and methods: Number of patients: 201 (85 males, 116 females), mean age 44.1 years. The US examinations were done by using a B-K Medical Hawk 2102 EXL scanner, the bioimpedance measurements were made by Biospace InBody 720 body composition analyser. Results: The mean VFT values increase parallel with BMI: in patients with BMI