CHAPTER XIX

BIOPHYSICS AND STRUCTURAL BIOLOGY

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esearch in biophysics had an early start in India in the late nineteenth century when the renowned physicist J.C. Bose began his pioneering work on the behaviour of cells under external stimulii. He also invented many delicate and sensitive instruments. The best known among them is the crescograph for recording plant growth. Among the modern biophysicists of India, and indeed of the world, the name of G.N. Ramachandran stands out as the most outstanding scientist to have worked in Independent India. The structure of the fibrous protein collagen, proposed by him and Gopinath Kartha in the first half of the 1950s, has been an intellectual achievement of the highest order and has stood the test of time. The Ramachandran plot, devised in the early 1960s, still remains the simplest and the most commonly used descriptor and versatile tool for the validation of protein structures. His contributions to the foundations of crystallography have been immense and in the area of image reconstruction he made major contributions. He initiated and pursued conformational studies on all major biopolymers and laid the foundations of the currently thriving field of molecular modelling. He founded two renowned schools of biophysics and structural biology, one at Chennai and the other at Bangalore. Although Ramachandran left the field nearly a quarter of a century ago and died recently, his work continues to exert great influence on structural studies of biomolecules.

N.N. Dasgupta of Kolkata has been another pioneer in the field of biophysics in India. He constructed an electron microscope in the middle of the last century and, among other things, visualised the cholera phage DNA. Another leading scientist associated with the early development of biophysics in the country has been A.R. Gopal-Iyengar who, working at Mumbai, made outstanding contributions in the basic and the applied aspects of radiobiology, radiation biophysics, cellular biophysics and related areas. Both of them left behind flourishing schools of biophysics. The others involved in pioneering efforts in the 1950s and the 1960s include D.M. Bose, N.N. Saha, S.N. Chatterjee, R.K. Poddar (all from Kolkata), S.R. Bawa (Chandigarh), R.K. Mishra (Delhi) and K.S. Korgaonkar (Mumbai). In addition to the students and colleagues of the early pioneers of biophysics in the country, a substantial section of the present leaders of biophysics and structural biology in India entered the field through doctoral or postdoctoral studies at leading schools in different parts of the world. Thus, the biophysics and structural biology community in India today is a vibrant mosaic made up of different strands of traditions and expertise.

CURRENT STATUS

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oday, close to a hundred research groups distributed in different institutions in the country are engaged in research in the field of biophysics. Among them, they cover almost all aspects of bioPURSUIT AND PROMOTION OF SCIENCE

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Photo: M.R.N. Murthy

Space-filling model of sesbania mosaic virus (left) and physalis mottle virus (right). Sesbania mosaic virus has a smoother surface when compared to physalis mottle virus which displays prominent protrusions at the 5-fold symmetry axis.

physics, especially at the molecular level. The range of activities is so extensive that it is impossible to cover all of them in a brief summary and the fact that the boundaries of biophysics are too porous to be defined makes the task all the more difficult. The effort here, therefore, is to give a gist of current biophysics research in India without making any attempt to be comprehensive. The strongest component of biophysics research in the country, and perhaps in the world, is concerned with molecular biophysics which, in modern parlance, is called structural biology. The emphasis here would thus be naturally on structural biology. Proteins and Peptides: G.N. Ramachandran and his colleagues were pioneers in structural studies on proteins at the international level. Much of their work has been computational or theoretical. The most important approach to studies on protein structure involves biological macromolecular crystallography. Work in this area was initiated early in the 1980s at the Indian Institute of Science (IISc) Bangalore, and the Bhabha Atomic Research Centre, Bombay. The endeavour gathered momentum with 202

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the implementation of the Thrust Area Programme of the Department of Science & Technology (DST) in 1983, when Bangalore was identified as a national nucleus for the development of this research area in the country. Since then, particularly in the 1990s, a number of research groups in macromolecular crystallography came into being in different parts of India with support from DST, the Department of Biotechnology (DBT) and the Council for Scientific and Industrial Research (CSIR). There are currently a dozen such groups located at nine institutions. The Indian effort in this area has been well coordinated and has a significant international presence. The macromolecular crystallographic studies in India range across a wide spectrum. Crystallographic studies on lectins, in conjunction with biochemical and physico-chemical investigations, carried out in India has had considerable international impact. The structure analysis of two plant viruses has been among the landmarks in the development of structural biology in India while studies of lactoferrins from different sources constitute an important component, as do those on phospholipases

GOPALASAMUDRAM NARAYANA RAMACHANDRAN 1922-2001

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onsidered one of the founders of molecular biophysics, G. N. Ramachandran was also a crystallographer of international repute. He was fascinated by the arrangement of molecules in proteins and so decided to take up research in this field. His work on the collagen molecule brought him worldwide reknown in the scientific community. Ramachandran was born on October 8,1922 in Ernakulam district of Kerala. He studied for a Master's degree in electrical engineering in 1940. He received his D.Sc. from the University of Madras in 1948 and completed his Ph.D. from the University of Cambridge in 1949. In 1951, he went back to Cambridge, England, to work with Sir William Lawrence Bragg, the 1915 Nobel Laureate in physics, and W.A. Wooster, and returned home with another Ph.D. Ramachandran is credited with the establishment of the Department of Physics at the University of Madras in 1952. He began work on modeling collagen from fibre diffraction in 1952-53, when J.D. Bernal, who did pioneering work in X-ray crystallography, visited him in Madras and suggested that the structure of collagen was one of the greatest unsolved problems. In 1954, Ramachandran arrived at the triple helix model which had two hydrogen bonds per unit. Dr Francis Crick, co-discoverer of the structure of the DNA molecule, had independently deduced one hydrogen bond per collagen unit. More recent single crystal work has shown the result to be 1.5 hydrogen bonds per unit, a compromise between these two proposals. In 1968, he was made the Jawaharlal Nehru Fellow at the University of Madras. In 1971, the Indian Institute of Science in Bangalore invited Ramachandran to set up the Department of Molecular Biophysics. Soon he managed to raise the Department to an international status where research was carried out on several giant molecules of biological importance, for example, nucleic acids and polysaccharides. In the 1970s, he turned to problems of mathematical logic and proposed a new method for structural analysis in 1990. RamachandranÕs work with A. V. Lakshminarayanan has been acclaimed as the starting point of the CAT scan technique in radiography. Ramachandran who was honoured with the prestigious Ewald Prize in 1999 for his outstanding contribution in the field of crystallography, died in Chennai on April 7, 2001 after a long illness.

and xylanases. Carbonic anhydrase and its complexes have been extensively studied in the country and work on ribosome-inactivating protein, and proteolytic enzymes and their inhibitors deserve mention. Substantial contributions have emanated from India in structural approaches to molecular mimicry. Crystallographic and related studies on immunological systems are now gathering momentum. Yet another crystallographic contribution from India pertains to the hydration, plasticity and action of proteins. An exciting recent development in the area is the concerted effort on

proteins from Mycobacterium tuberculosis. Nuclear Magnetic Resonance (NMR) is widely used in chemistry and biology, and structure determination of proteins using NMR is currently gathering momentum in India. The structures of a couple of proteins, a calcium-binding protein and a novel neurotoxin, have already been determined at the Tata Institute of Fundamental Research, Mumbai, which houses the largest national NMR facility in the country. Serious efforts in the area are now underway in a few more centres in the country. In addition to working on individual or PURSUIT AND PROMOTION OF SCIENCE

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Photo: N.R. Jagannathan

groups of molecules, the Mumbai centre and the NMR centre at the IISc, Bangalore, which houses a widely used national facility, have made important contributions to the methodology of 2D and 3D NMR spectroscopy. Computational approaches to protein structure continues to be an important component of biophysical research in India. The work in the area encompasses homology modelling, protein-ligand

Transaxial T1-weighted proton MR image of a patient showing clearly the tumor near the 3rd ventricle region of the brain.

interactions, study of secondary and other special structural features, and data analysis. Biophysical chemistry is central to modern biology. There was a phase in India when studies in this area appeared to be on the decline but in recent years, work in the area has gathered great momentum, particularly in relation to the folding, stability and design of proteins. High-quality work of international standard on protein folding is 204

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currently being carried out in several laboratories in India. Some of them primarily address general questions, while others tend to concentrate on particular systems. The systems encompass proteins from widely different families. Protein stability, to a substantial degree, is related to protein folding. This is reflected in the work carried out in many laboratories. The role of water, metal ions, ligands and additives in protein stability also form a topic of detailed investigations. Molecular design provides a link between proteins and peptides. Design and synthesis of oligopeptides with desired conformations, particularly using conformationally restrictive amino acids, have been an area of considerable strength in the country. Such peptides are then used as modular units for the subsequent synthesis of proteins. Imaginative use of crystallography and NMR has played an important part in this effort. Interactions involving amino acids and peptides and their relevance to present-day biological systems, as well as to processes involved in the origin of life, have been investigated in considerable detail. Synthetic, spectroscopic, crystallographic and theoretical studies have also been carried out on several peptide systems. Nucleic Acids: Theoretical conformational studies on nucleic acids were initiated by G.N. Ramachandran and were carried forward by V. Sasisekharan and his colleagues. In structural studies, using crystallography, on DNA, M.A. Viswamitra has been another pioneer. Computational and crystallographic studies, particularly on the sequence-dependent structure of DNA, continue to be pursued seriously in several laboratories. Extensive work on the structure determination of oligonucleotides using NMR has emerged, particularly from the TIFR, Mumbai. Work at this centre has produced a wealth of information on unusual DNA structures. In the process, important contributions to NMR methodology specifically applicable to DNA, have

The unusual quarternary structure of peanut lectin (left) and a subunit of jacaline which exhibits a novel lectin fold (right).

also been made. DNA-ligand interactions are pursued vigorously, mainly using spectroscopic and physico-chemical techniques. The recently recognized importance of trinucleotide and other repeats has added a new dimension to the structural studies on DNA. Membranes: The biological membrane, the ubiquitous multimolecular system found in living organisms, and related models, have received considerable attention from biophysicists. India has been no exception in this regard. The organization, heterogeniety, dynamics and asymmetry of membranes have been studied extensively, using spectroscopic and physico-chemical approaches. The Indian contribution towards the synthetic, spectroscopic, crystallographic and computational

studies on ionophores has been very substantial. The same is true about the interaction of drugs and other active molecules with the membrane. Significant contributions have also been recently made from India to ion channels with implications, among other things, for neurobiology. Carbohydrates: Carbohydrates have emerged as very interesting molecules during the past couple of decades. A substantial part of biological recognition is mediated by sugars. Work on proteins that bind sugars such as lectins, has already been referred to. Pioneering efforts of V.S.R. Rao and his colleagues on polysaccharide conformation have been noteworthy. Yet, work on carbohydrates and glycoconjugates in the country is much less extensive than it ought to be, although there are some groups working in the area. Computational Biology and Bioinformatics: Indians have been in the forefront of international efforts in computational biology during the emergent phase of PURSUIT AND PROMOTION OF SCIENCE

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the field and work in the field continues to be pursued vigorously in the country. Some components of it have been referred to above. There are groups of scientists, distributed over the country in different centres, who are exclusively concerned with computational biology. Thanks to a major initiative of DBT, India has been among the first countries to embark on a concerted effort in the allied field of bioinformatics. A network of bioinformatics centres have been established covering all regions of the country for the dissemination and analysis of different data bases. This network has played an invaluable role in taking bioinformatics to the biology community as a whole. The emphasis of the bioinformatics programme is shifting from dissemination to generation of data bases and software. Whole Systems: Structural biology has to some extent dominated biophysics in recent years. However, biophysical studies at higher levels of organization continue to be pursued as for instance in NMR investigations of whole cells and metabolic processes and, importantly, in MRI studies on neurological processes. Optical studies of different types on biological systems are being carried out in a few centres. Radiation biophysics is another area in which significant activity exists while work in medical biophysics and on biomaterials is being pursued at a few centres. Unlike in the case of computational biophysics, mathematical biology has had only limited following in the country. However, a trend towards wider acceptance of the area is now discernible. Theoreticians, particularly theoretical physicists, are currently showing increased interest in the mathematical modeling of biological systems.

TRENDS AND PROSPECTS

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ven in normal circumstances, predictions rarely come true in science. This is even more so at present when rapid and revolutionary changes are taking place in almost all branches of biology. All that one can reasonably do is to indicate some trends. For long, much of the biophysics research in 206

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India, impressive though it was, took place independently of the bulk of the biological research activities in the country. Work in crystallography and computational biology exemplifies the situation. Most of the workers in these areas used to be physicists and had little professional contact with biologists. The situation is rapidly changing. A number of multi-disciplinary collaborations have developed. Structural biologists are now very much part of the biology community and biology laboratories are rapidly becoming natural habitats of crystallographers and computational biologists. This trend is likely to accelerate in the years to come. The development of biological macromolecular crystallography in India provides a good example of a concerted effort towards building up momentum in an important scientific programme in the country. The area of biological macromolecular crystallography is well past the stage of capacity building and many important problems have already been successfully addressed at a globally competitive level. Work using the crystallographic approach is poised to take further major strides, with the active involvement of biologists working in other areas. A further impetus in this direction is expected to be provided by the recently evolved multi-institutional programme on the structural genomics of microbial pathogens. As indicated earlier, macromolecular structure determination using NMR is well underway in one major centre in the country and major results in the area are expected in the near future, from at least two more centres. However, in experimental approaches to macromolecular structure determination, a major gap area in the country is cryoelectron microscopy. Efforts to fill this gap will be very worthwhile. The major thrust of biological structural studies in India has been concerned with proteins that act on carbohydrates, plant viruses, lactotransferrins, proteolytic enzymes and their inhibitors, protein hydration, molecular mimicry and structural variability of DNA. These lines of investigation are expected to be further

strengthened. Work on immunologically relevant problems, which has already been initiated, is likely to gather additional momentum in the near future. A major new thrust is expected in structural studies on proteins from microbial pathogens. A major theme of structural studies on peptides has been concerned with protein folding and design. Physico-chemical, thermodynamic, structural and theoretical studies in this area will continue to be an important element of structural biology in the country while biophysical and theoretical approaches should continue to play a major role in multifarious studies on membranes. The primary emphasis of the bioinformatics programme in India so far has been on the dissemination of information. That phase is now over. Efforts are already underway for the creation of novel data bases and the development of webbased software. These efforts, which are now gathering momentum, are likely to occupy centre stage of bioinformatics in the country in the future. The initiatives in genome analysis have added a new dimension to these efforts. In general, with the departure of giants of yesteryears, the visibility of computational biology as a distinct approach, diminished somewhat during the closing decades

of the twentieth century. This trend is now being reversed as a new generation of practitioners in the area have appeared and seem to be set to make a significant impact on biology. Work on whole systems, particularly using imaging techniques, is expected to be further carried forward in the years to come. There is currently a clearly discernible and welcome trend towards harmonizing studies at the molecular and the organismal levels. This trend needs to be, and is likely to be, strengthened in the future. Recent years have witnessed the rapid evolution of a host of new approaches and novel techniques to deal with biology at different levels of organization. Most of them involve a great deal of what is conventionally described as biophysics. However, the borders between different subdisciplines of biology are becoming increasingly blurred. Studies at different levels of biological organization have also begun to get progressively integrated. In this rapidly changing scenario, it is difficult to foretell how the new approaches and techniques will develop in the country. However, modern biological research in India is sufficiently mature and resilient to be able to cope with the rapid developments and contribute to them.

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