Three-dimensional dynamic structure of lipid bilayer membranes saturated with cholesterol. Promising advances at Q-band and W-band Witold Karol Subczynski Department of Biophysics Medical College of Wisconsin
Acknowledgements •
Collaborators:
•
Marija Raguz, MSc, Medical College of Wisconsin (graduate student)
•
Justyna Widomska, PhD and Anna Wisniewska, PhD, Jagiellonian University (postdocs)
•
James S. Hyde, PhD, Medical College of Wisconsin
•
Akihiro Kusumi, PhD, DSc, Kyoto University
•
Funded by NIH grants EY015526 and TW008052
DMPC/cholesterol phase diagram The broken lines indicate the DMPC/cholesterol mixtures and temperatures mainly addressed in our work.
Lipid Rafts in Eye Lens: Discrimination by Pulse EPR
• 9 mm in diameter and 4 mm thick, contains 1 000–3 000 layers of fiber cells. • To prevent excessive light scattering and compromised lens transparency, fiber cells loss all subcellular organelles during maturation. • Due to minimal cell turnover, cells in the nucleus of the human lens may be considered as the longest-lived cells in our body, as old as the individual. • The fiber cell plasma membrane become essentially the only supramolecular structure of the adult lens.
Fiber-cell plasma membrane • •
•
Overloaded with cholesterol, which saturates phospholipid bilayer and forms cholesterol crystalline domains (CCDs, two-dimensional cholesterol crystals) within these membranes. The appearance of CCDs is usually a sign of pathology; however, only in the eye lens cholesterol crystalline domains play a positive physiological function, maintaining lens transparency and possibly protecting against cataract formation. Cholesterol biosynthesis inhibiting drugs causes cataract formation in the rat, dog, and human.
•
Cholesterol in the lens plays an important physiological role.
•
Better understanding of cholesterol functions on a molecular level.
Age-related trends in cholesterol content of human lenses • The changes manifest themselves as an increase of the total cholesterol-to-phospholipid mole ratio and as a higher cholesterol-to-phospholipid mole ratio in the nucleus as compared with the cortex. • Animals with long lifespan exhibit higher cholesterol-tophospholipid mole ration than animals with short lifespan. • Also phospholipid composition of the eye lens membranes change significantly with ageing, between regions of the lens, and between animal species. In contrast, there are no major differences in the lipid composition of most organs from one species to another and with age.
Relative abundance of phospholipid classes in animal lenses crude lipid extracts • aaa
SM(d18:1) sphingomyelin SM(d18:0) dihydrosphingomyelin GPCho phosphatidylcholine GPEtn phosphatidylethanolamine GPSer phosphatidylserine The phospholipid composition of adult human lens membranes (60 year old human) differs dramatically from that of any other mammalian membrane.
The need for a high cholesterol content in the lens is unclear • Due to minimal cell turnover, cells in the nucleus of the human lens may be considered as the longest-lived cells in human body. • I will present data which support our main hypothesis, that the high cholesterol content and the presence of the cholesterol crystalline domain are needed to maintain lens membrane homeostasis through the life of the individual.
Spin labels • In these studies, phospholipidtype spin labels, as well as cholesterol analogues, are introduced to the investigated membranes. Because of the overall similarity of the molecular structure of these spin labels with phospholipids and cholesterol; they should be distributed between different membrane domains similarly to the distribution of phospholipids and cholesterol.
S = 0.5407 ( AII' − A⊥' ) / a0
Profiles of the order parameter
•
The order parameter, S = 0.5407(A’II – A’L)/a0 where a0 = (A’II + A’L)/3 , is a measure of the amplitude of the wobbling motion. Increase in order parameter indicates that the angle of the cone, responsible for the wobbling motion of the alkyl chain, decreases. Although the order parameter indicates the static property of the lipid bilayer, the change in the order parameter is most often described as a change in spinlabel mobility, and thus as a change in membrane fluidity.
Fluidity profiles
There are no easily obtainable EPR spectral parameters for lipid spin labels that can describe profiles of membrane fluidity related to dynamic membrane properties. The spin-lattice relaxation time (T1) in the deoxygenated samples depends primarily on the rate of motion of the nitroxide moiety within the lipid bilayer, and thus describes the dynamics of the membrane environment at the depth at which the nitroxide fragment is located. This spectral parameter can be obtained from SR EPR measurements with lipid spin labels. Thus, T1 can be used as a convenient quantitative measure of membrane fluidity that indicates the rate of motion of phospholipid alkyl chains (or nitroxide free radical moieties attached to those chains).
Hydrophobicity profiles
The hydrophobicity profiles across lipid-bilayer membranes is primarily monitored using AZ (z-component of the hyperfine-interaction tensor of the nitroxide spin labels) as a convenient experimental observable. AZ can be immediately obtained from EPR spectra of spin labels measured from the frozen suspension of membranes (A). With an increase in solvent polarity, AZ increases. In this type of work, a nitroxide free-radical moiety is placed at various depths in the lipid bilayer, and hydrophobicity profiles across membranes are obtained (see Fig. B). In our work, we related hydrophobicity, as observed by AZ at a selected depth in the membrane (B), to hydrophobicity (or ε) of bulk organic solvent by referring to Fig. C.
Profiles of the oxygen transport parameter
W(x) = T1-1(Air, x) - T1-1(N2, x), T1 is the spin-lattice relaxation in samples equilibrated with atmospheric air and nitrogen, respectively.
W(x) = AD(x)C(x),
A = 8πpr0,
where r0 is the interaction distance between oxygen and the nitroxide moiety of the spin label (4.5 Å) and p is the probability that an observable event occurs when a collision does occur. C(x) and D(x) are, respectively, the local oxygen concentration and the local oxygen diffusion coefficient at a "depth" x in the membrane that is in equilibrium with atmospheric air:
Discrimination by Oxygen Transport
•
•
When located in two different membrane domains, the spin label alone most often cannot differentiate between these domains. However, even small differences in lipid packing in these domains will affect oxygen partitioning and oxygen diffusion, which can be easily detected by the different T1 values. The saturation-recovery signal is a simple double-exponential curve with time constants of: – T1-1 (air, SLOT) and T1-1 (air, FOT)
The distribution and approximate locations of lipid spin labels in domains formed in phospholipid membranes with different cholesterol contents. (A) Phospholipid spin labels (5-, 10-, 16-, and TPC, and 9-SASL) are distributed uniformly within the phospholipid bilayer without cholesterol. (B) When the cholesterol content is close to the solubility threshold (~50 mol%), the membrane should be in the liquid-ordered-like phase; separate domains in this case are not expected, and all spin labels should be uniformly distributed within the bilayer. (C) Phospholipid spin labels are only located in the bulk phospholipidcholesterol domain when it coexists with the pure cholesterol crystalline domain for cholesterol content exceeding the solubility threshold; however, spin-labeled cholesterol analogues are distributed between both domains.
Profiles of the Hydrocarbon Chain Order
Hydrophobicity Profiles
Profiles of the Oxygen Transport Parameter
Saturating amount of cholesterol forms hydrophobic and rigidity barriers and creates hydrophobic channels
The hydrophobicity in the membrane center is comparable to that in hexane and hexadecane (epsilon from 2 to 3). This greatly increases the activation energy required for polar and ionic small molecules to pass the membrane. Also it forms the resistance to permeation of small hydrophobic molecules across the membrane located at the polar headgroup region and the nearsurface region in the hydrocarbon phase, called the rigidity barrier. The resistance of the membrane center to the lateral transport of small hydrophobic molecules is much lower than in water phase and the solubility of small hydrophobic molecules is much higher. We call this region “hydrophobic channels.”
Cholesterol decreases the vertical fluctuations of all membrane components
The change from the gel phase-like membrane to the liquid crystalline-like membrane occurs between the C9 and C10 within the distance of one carbon-carbon bond (i.e. 1.3-1.5 Å) along the alkyl chain. This transition is smooth for membranes without cholesterol or membranes containing a small concentration of cholesterol. It indicates that high cholesterol content decreases the vertical fluctuations of all membrane components, so that the rigid plate-like portion of all cholesterol molecules are aligned at the same depth of the C9 in the alkyl chains. We hypothesize that this alignment help to decrease light scattering at the membrane and, thus, helps to maintain lens transparency.
CCD saturates surrounding membranes with cholesterol • The lipid composition of the lens fiber-cell membrane changes as both animals and humans aged. Usually, such notable change would result in alteration of the membrane’s physical properties, which would affect the function of proteins immersed in the lipid bilayer. • We hypothesize that the extremely high (saturating) content of cholesterol in the fiber-cell membrane keeps the bulk physical properties of the lipid-bilayer portion of the membrane consistent and independent of changes in the phospholipid composition. • The CCD has some function specific to the fiber-cell membrane. The CCD provides buffering capacity for cholesterol concentration in the surrounding phospholipid bilayer, keeping it at a constant saturating level. • The CCD has a crucial role in maintaining homeostasis of the lens membrane, the only supramolecular structure of matured fiber cells.
Phospholipid composition controls formation of the CCD Phase diagram for phospholipid/cholesterol membranes based on the cholesterol solubility thresholds in PS, PC, and SM membranes. Y-axis indicates the pure phospholipid (arrows) and mixtures of phospholipids (between arrows). It is assumed that the cholesterol solubility threshold value in the phospholipid mixture is a weighted sum of individual thresholds with a weight equal to the mol fraction of the individual phospholipid in the mixture. ld, lo, and CCD indicate liquid-disordered, liquid-ordered and cholesterol crystalline domains, respectively. The solid line indicates the cholesterol-to-phospholipid mixing ratio above which the CCD should be formed.
Phospholipid composition controls formation of the CCD
The threshold of the cholesterol solubility differs significantly in a phospholipid membranes being 1/2, 1/1, 1/1, and 2/1 for Chol/PS, Chol/PC, Chol/PE, and Chol/SM, respectively
The relationship between cholesterol solubility thresholds in lens membranes and maximum lifespan for different species. The cholesterol solubility thresholds (above this cholesterol concentration CCDs should be formed) were evaluated based on the phospholipid compositions and the assumption that the cholesterol solubility threshold value in the phospholipid mixture is a weighted sum of individual thresholds with a weight equal to the mol fraction of the individual phospholipid in the mixture. Points are for mouse (3), rat (4), chick (6), sheep (20), pig (23), cow (30), and human (70). In parentheses are values for maximum lifespan.
Phospholipid composition controls formation of the CCD Thus, in animals with a long lifespan, lens membrane phospholipid composition ensures formation of CCDs at significantly higher cholesterol concentration than in animals with a short lifespan In humans and animals, the lens phospholipid composition changes with age that way which delays formation of CCDs. The relationship between cholesterol solubility thresholds in lens membranes and the age of the human donor.
Cholesterol content in lens increases also with age reaching value of 4 in nucleus of aged lenses
Spin label T1 values from 2 to 94 GHz
The X-band SR spectrometer is equipped with a loop-gap resonator (with a sample volume of ~3 μl). A new Q-band and W-band SR instruments have been built and new loop-gap resonators have sample volumes of ~30 nl. T1 values of spin labels in water samples and in membranes increase when microwave frequency increases, reaching maximum at Q-band (35 GHz), and decrease when measured at W-band (94 GHz) These new capabilities at Q-band have the potential to be an extremely powerful tool for studying membrane domains.
Oxygen transport parameter from 2 to 94 GHz Freq.(GHz)
S1 (2.54) S2 (2.54) X (9.2)
K (18.5)
Q (34.6)
W (94.0)
T1 (μs)
0.69
0.94
2.54
3.46
3.69
2.05
W (μs-1)
2.68
2.71
2.67
2.54
2.68
3.02
The effect of bimolecular collisions of oxygen on observed spin label relaxation rates is independent of frequency. W(x) = T1-1(Air, x) - T1-1(N2, x), Spin label oximetry is a widely-used technique in SDSL and DOT.
Saturation-recovery at Q-band
Capabilities of the Q-band SR EPR spectrometer equipped with the three-loop–twogap resonator with a sample volume of 30 nl. (A,B) Q-band SR signals of 5-PC in POPC membranes obtained in the absence and presence of molecular oxygen showing good signal-to-noise ratio and excellent fit to single exponentials (see residuals). (C) Profile of the oxygen transport parameter across the POPC membrane obtained at Qband. The profile is practically the same as those obtained at X-band with the use of the loop-gap resonator with a sample volume of 3 μl.
Promising advances at Q-band and W-band •
• • • •
• •
• •
T1 values of spin labels have maximum at Q-band (35 GHz).
The oxygen transport parameter measured in the aqueous phase and in membranes is independent of microwave frequency. We conclude that the longest values of T1 will generally be found at Q-band, noting that long values are advantageous for measurement of bimolecular collisions with oxygen or paramagnetic metal ions. These benefits, together with a small sample volume and other factors such as a higher resonator efficiency parameter and a new technique for canceling free induction decay contamination, are promising in using Q-band SR. All profiles obtained at X-band for calf- and pig-lens lipid membranes were obtained for samples prepared from 50 eyes. Similarly, measurements for cortical and nuclear cow-lens lipid membranes were based on samples prepared from 100 eyes. Because sample volume at Q-band is 100 times smaller, reliable profiles can be obtained based on samples prepared from one eye. We have become proficient in handling, degassing, and equilibrating with gas mixture samples of this size. Yin and Hyde showed that the use of high observing power in SR EPR experiments does not affect the ability to extract bimolecular collision rates and can increase the signal-to-noise ratio up to ten times. Thus, we will use this approach if the signal-to-noise ratio in samples prepared from one eye is too low. Dr. Hyde aim to build an improved loop-gap resonator and optimize liquid-phase concentrationsensitivity for spin labels at Q-band, that will increase EPR sensitivity by a factor of 5. If we face sensitivity problems at Q-band, they will be solved using the SR W-band spectrometer, which has about three-times greater sensitivity (because of the Boltzmann equilibrium) for samples of the same volume. In addition, it offers the same sensitivity for samples with twenty-times smaller volume using the W-band loop-gap resonator.
Cholesterol crystalline domain function in eye lens: EPR spin-labeling studies The broad objective of this proposal is to understand cholesterol’s function in the human eye lens, focusing on the cholesterol crystalline domain formed in fiber-cell plasma membranes. The proposed studies will generate important fundamental information about topographical and age-related differences in cholesterol-membrane interactions in the eye lens that should increase our understanding of the role cholesterol plays and, in turn, help contribute to the prevention of age-related nuclear cataracts. I would like to invite a postdoc and/or PhD student to work with me on this grant, which will start on December 1, 2009 and end on November 30, 2014. E-mail:
[email protected] Phone: 414 456 4038
Thank you
Slide show design by MAS
