Although the importance of the polyelectrolyte character of DNA has been recognized for some time (Felsenfeld & Miles 1967), few of the implications have been explored, primarily because of a lag in translating the breakthroughs in polyelectrolyte theory of the last decade into a form that is well adapted to the analysis of the specialized problems of biophysical chemistry. Perhaps an analogous situation existed in the field of protein chemistry during the period after the formulation and confirmation of the Debye—Hückel theory of ionic solutions but before Scatchard's incorporation of the theory into his analysis of the binding properties of proteins. An achievement for polynucleotide solutions parallel to Scatchard's was recently presented by Record, Lohman, & de Haseth (1976) and further developed and reviewed by Record, Anderson & Lohman (1978).

Cryo-electron microscopy of vitrified specimens was just emerging as a practical method when Richard Henderson proposed that we should teach an EMBO course on the new technique. The request seemed to come too early because at that moment the method looked more like a laboratory game than a useful tool. However, during the months which ellapsed before the start of the course, several of the major difficulties associated with electron microscopy of vitrified specimens found surprisingly elegant solutions or simply became non-existent. The course could therefore take place under favourable circumstances in the summer of 1983. It was repeated the following years and cryo-electron microscopy spread rapidly. Since that time, water, which was once the arch enemy of all electronmicroscopists, became what it always was in nature – an integral part of biological matter and a beautiful substance.

1. Introduction 370

2. Infrared (IR) spectroscopy – general principles 372

2.1 Vibrations 372

2.2 Information that can be derived from the vibrational spectrum 372

2.3 Absorption of IR light 375

3. Protein IR absorption 376

3.1 Amino-acid side-chain absorption 376

3.2 Normal modes of the amide group 381

4. Interactions that shape the amide I band 382

4.1 Overview 382

4.2 Through-bond coupling 383

4.3 Hydrogen bonding 383

4.4 Transition dipole coupling (TDC) 383

5. The polarization and IR activity of amide I modes 387

5.1 The coupled oscillator system 387

5.2 Optically allowed transitions 388

5.3 The infinite parallel β-sheet 388

5.4 The infinite antiparallel β-sheet 389

5.5 The infinite α-helix 390

6. Calculation of the amide I band 391

6.1 Overview 391

6.2 Perturbation treatment by Miyazawa 393

6.3 The parallel β-sheet 394

6.4 The antiparallel β-sheet 395

6.5 The α-helix 396

6.6 Other secondary structures 398

7. Experimental analysis of protein secondary structure 398

7.1 Band fitting 398

7.2 Methods using calibration sets 401

7.3 Prediction quality 403

8. Protein stability 404

8.1 Thermal stability 404

8.2 1H/2H exchange 406

9. Molecular reaction mechanisms of proteins 408

9.1 Reaction-induced IR difference spectroscopy 408

9.2 The origin of difference bands 409

9.3 The difference spectrum seen as a fingerprint of conformational change 410

9.4 Molecular interpretation: strategies of band assignment 416

10. Outlook 419

11. Acknowledgements 420

12. References 420

This review deals with current concepts of vibrational spectroscopy for the investigation of protein structure and function. While the focus is on infrared (IR) spectroscopy, some of the general aspects also apply to Raman spectroscopy. Special emphasis is on the amide I vibration of the polypeptide backbone that is used for secondary-structure analysis. Theoretical as well as experimental aspects are covered including transition dipole coupling. Further topics are discussed, namely the absorption of amino-acid side-chains, 1H/2H exchange to study the conformational flexibility and reaction-induced difference spectroscopy for the investigation of reaction mechanisms with a focus on interpretation tools.

Starting from known properties of non-specific salt effects on the surface tension at an air–water interface, we propose the first general, detailed qualitative molecular mechanism for the origins of ion-specific (Hofmeister) effects on the surface potential difference at an air–water interface; this mechanism suggests a simple model for the behaviour of water at all interfaces (including water–solute interfaces), regardless of whether the non-aqueous component is neutral or charged, polar or non-polar. Specifically, water near an isolated interface is conceptually divided into three layers, each layer being 1 water-molecule thick. We propose that the solute determines the behaviour of the adjacent first interfacial water layer (I1); that the bulk solution determines the behaviour of the third interfacial water layer (I3), and that both I1 and I3 compete for hydrogen-bonding interactions with the intervening water layer (I2), which can be thought of as a transition layer. The model requires that a polar kosmotrope (polar water-structure maker) interact with I1 more strongly than would bulk water in its place; that a chaotrope (water-structure breaker) interact with I1 somewhat less strongly than would bulk water in its place; and that a non-polar kosmotrope (non-polar water-structure maker) interact with I1 much less strongly than would bulk water in its place.

We introduce two simple new postulates to describe the behaviour of I1 water molecules in aqueous solution. The first, the ‘relative competition’ postulate, states that an I1 water molecule, in maximizing its free energy (—δG), will favour those of its highly directional polar (hydrogen-bonding) interactions with its immediate neighbours for which the maximum pairwise enthalpy of interaction (—δH) is greatest; that is, it will favour the strongest interactions. We describe such behaviour as ‘compliant’, since an I1 water molecule will continually adjust its position to maximize these strong interactions. Its behaviour towards its remaining immediate neighbours, with whom it interacts relatively weakly (but still favourably), we describe as ‘recalcitrant’, since it will be unable to adjust its position to maximize simultaneously these interactions. The second, the ‘charge transfer’ postulate, states that the strong polar kosmotrope–water interaction has at least a small amount of covalent character, resulting in significant transfer of charge from polar kosmotropes to water–especially of negative charge from Lewis bases (both neutral and anionic); and that the water-structuring effect of polar kosmotropes is caused not only by the tight binding (partial immobilization) of the immediately adjacent (I1) water molecules, but also by an attempt to distribute among several water molecules the charge transferred from the solute. When extensive, cumulative charge transfer to solvent occurs, as with macromolecular polyphosphates, the solvation layer (the layer of solvent whose behaviour is determined by the solute) can become up to 5- or 6-water-molecules thick.

We then use the ‘relative competition’ postulate, which lends itself to simple diagramming, in conjunction with the ‘charge transfer’ postulate to provide a new, startlingly simple and direct qualitative explanation for the heat of dilution of neutral polar solutes and the temperature dependence of relative viscosity of neutral polar solutes in aqueous solution. This explanation also requires the new and intriguing general conclusion that as the temperature of aqueous solutions is lowered towards o °C, solutes tend to acquire a non-uniform distribution in the solution, becoming increasingly likely to cluster 2 water molecules away from other solutes and surfaces (the driving force for this process being the conversion of transition layer water to bulk water). The implications of these conclusions for understanding the mechanism of action of general (gaseous) anaesthetics and other important interfacial phenomena are then addressed.

The purpose of this review is to examine the various effects of low- molecular-weight electrolytes on the associations and interactions of proteins and nucleic acids. Our primary interest is in general electrostatic effects, rather than chemical effects (specific interactions) of particular ions (e.g. transition metals, protons). We consider those interactions in which a variation in salt concentration has a significant effect on the macromolecular equilibrium, and analyse the effects of salt in these situations in terms of (i) direct participation of ions in the biopolymer reaction, (ii) Debye—Hückel screening by salt ions of the charge interactions on the biopolymers, and (iii) the reduction in water activity brought about at high salt concentrations.

Membranes are the most common cellular structures in both plants and animals. They are now recognized as being involved in almost all aspects of cellular activity ranging from motility and food entrapment in simple unicellular organisms, to energy transduction, immunorecognition, nerve conduction and biosynthesis in plants and higher organisms. This functional diversity is reflected in the wide variety of lipids and particularly of proteins that compose different membranes. An understanding of the physical principles that govern the molecular organization of membranes is essential for an understanding of their physiological roles since structure and function are much more interdependent in membranes than in, say, simple chemical reactions in solution. We must recognize, however, that the word ‘understanding’ means different things in different disciplines, and nowhere is this more apparent than in this multidisciplinary area where biology, chemistry and physics meet.

Though the structures presented in crystallographic models of macromolecules appear to possess rock-like solidity, real proteins and nucleic acids are not particularly rigid. Most structural work to date has centred upon the native state of macromolecules, the most probable macromolecular form. But the native state of a molecule is merely its most abundant form, certainly not its only form. Thermodynamics requires that all other possible structural forms, however improbable, must also exist, albeit with representation corresponding to the factor exp( — Gi/RT) for each state of free energy Gi (see Moelwyn-Hughes, 1961), and one appreciates that each molecule within a population of molecules will in time explore the vast ensemble of possible structural states.

Proton and carbon-13 nmr spectra of unsonicated lipid bilayers and biological membranes are generally dominated by strong proton–proton and proton–carbon dipolar interactions. As a result the spectra contain a large number of overlapping resonances and are rather difficult to analyse. Nevertheless, important information on the structure and dynamic behaviour of lipid systems has been provided by these techniques (Wennerström & Lindblom, 1977).

Crystallography supplies unparalleled detail on structural information critical for mechanistic analyses; however, it is restricted to describing low energy conformations of macromolecules within crystal lattices. Small angle X-ray scattering (SAXS) offers complementary information about macromolecular folding, unfolding, aggregation, extended conformations, flexibly linked domains, shape, conformation, and assembly state in solution, albeit at the lower resolution range of about 50 Å to 10 Å resolution, but without the size limitations inherent in NMR and electron microscopy studies. Together these techniques can allow multi-scale modeling to create complete and accurate images of macromolecules for modeling allosteric mechanisms, supramolecular complexes, and dynamic molecular machines acting in diverse processes ranging from eukaryotic DNA replication, recombination and repair to microbial membrane secretion and assembly systems. This review addresses both theoretical and practical concepts, concerns and considerations for using these techniques in conjunction with computational methods to productively combine solution scattering data with high-resolution structures. Detailed aspects of SAXS experimental results are considered with a focus on data interpretation tools suitable to model protein and nucleic acid macromolecular structures, including membrane protein, RNA, DNA, and protein–nucleic acid complexes. The methods discussed provide the basis to examine molecular interactions in solution and to study macromolecular flexibility and conformational changes that have become increasingly relevant for accurate understanding, simulation, and prediction of mechanisms in structural cell biology and nanotechnology.

Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associatedenergy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of ˜ 105 molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10000 particles are required. Below 105 molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

SUMMARY 241

1. INTRODUCTION 242

1.1 Milestones in the history of HS 244

1.2 Solvation forces 245

2. PHENOMENOLOGY OF THE HOFMEISTER SERIES 246

2.1 Extension of the properties of HS from inorganic to organic cosolutes 246

2.2 Effect of salts on the stability of biomolecules 247

2.3 Effect on enzyme activity 248

2.4 Influence of salts on the crystallization of biological macromolecules 249

2.5 Effects of salts on DNA–protein interactions 250

2.6 Interactions at receptor sites and effects on ion channels 251

2.7 Water-coordinating properties of salts as determined by gel chromatographic behaviour 254

3. INTERFACIAL FREE ENERGY 254

3.1 Types of forces 254

3.2 Distance dependence of the interactions 257

4. THE NATURE OF SURFACES AND LIQUIDS 259

4.1 Proteins 259

4.2 Adsorbing surfaces 260

4.3 Water 261

5. THE EFFECT OF COSOLUTES ON WATER 263

5.1 Electrochemistry 263

5.2 Neutron scattering 264

5.3 Vibrational spectroscopy 265

5.4 Nuclear magnetic resonance 265

6. COSOLUTE EXCLUSION FROM SURFACES 266

6.1 Cosolute enrichment 268

7. CONCLUSIONS AND OUTLOOK 268

8. ACKNOWLEDGEMENTS 269

9. REFERENCES 269

A. GLOSSARY 276

B. LIST OF PRINCIPAL SYMBOLS 276

Advances in experimental and computational methodologies have led to a recent renewed interest in the Hofmeister series and its molecular origins. New results are surveyed and assessed. Insights into the underlying mechanisms have been gained, although deeper molecular understanding still seems to be elusive. The principal reason appears to be that the Hofmeister series emerges from a combination of a general effect of cosolutes (salts, etc.) on solvent structure, and of specific interactions between the cosolutes and the solute (protein or other biopolymer). Hence every system needs to be studied individually in detail, a state of affairs which is likely to continue for some time. A deeper understanding of the Hofmeister series can be an extraordinarily valuable guide to designing experiments, including not only those probing the series per se, but also those designed to elucidate the adsorption, aggregation and stabilization phenomena which underlie so many biological events. The aim of this review is to provide an up-to-date framework to guide such understanding, consolidating recent advances in the many fields on which the Hofmeister series impinges.

Correlating the structure and action of biological molecules requires knowledge of the corresponding relation between structure and energy. Probably the most important factors in such a structure– energy correlation are associated with electrostatic interactions. Thus the key requirement for quantative understanding of the action of biological molecules is the ability to correlate electrostatic interactions with structural information. To appreciate this point it is useful to compare the electrostatic energy of a charged amino acid in a polar solvent to the corresponding van der Waals energy. The electrostatic free energy, ΔGel, can be approximated (as will be shown in Section II) by the Born formula (ΔGel = –(166Q2/ā) (I – I/E)). Where ΔGel is given in kcal/mol, Qis the charge of the given group, in units of electron charge, āis the effective radius of the group, and E is the dielectric constant of the solvent. With an effective radius of charged amino acids of approximately 2 Å, Born's formula gives about – 80 kcal/mol for their energy in polar solvents where E is larger than 10. This energy is two orders of magnitude larger than the van der Waals interaction of such groups and their surroundings.

Alcohol based cosolvents, such as trifluoroethanol (TFE) have been used for many decades to denature proteins and to stabilize structures in peptides. Nuclear magnetic resonance spectroscopy and site directed mutagenesis have recently made it possible to characterize the effects of TFE and of other alcohols on polypeptide structure and dynamics at high resolution. This review examines such studies, particularly of hen lysozyme and β-lactoglobulin. It presents an overview of what has been learnt about conformational preferences of the polypeptide chain, the interactions that stabilize structures and the nature of the denatured states. The effect of TFE on transition states and on the pathways of protein folding and unfolding are also reviewed. Despite considerable progress there is as yet no single mechanism that accounts for all of the effects TFE and related cosolvents have on polypeptide conformation. However, a number of critical questions are beginning to be answered. Studies with alcohols such as TFE, and ‘cosolvent engineering’ in general, have become valuable tools for probing biomolecular structure, function and dynamics.

1. COSOLVENTS: OLD HAT? 298

2. HOW DOES TFE WORK? 299

2.1 Effect on hydrogen bonding 300

2.2 Effect on non-polar sidechains 301

2.3 Effect on solvent structure 302

3. EFFECTS OF TFE ON (UN-)FOLDING TRANSITIONS 303

3.1 Pretransition 303

3.2 Transition 304

3.3 Posttransition 305

3.4 Far UV CD spectroscopic detection of structure 306

3.5 Effect with temperature 306

3.6 Effect with additional denaturants 306

4. THERMODYNAMIC PARAMETERS FROM STRUCTURAL TRANSITIONS OF PEPTIDES AND PROTEINS IN TFE 307

5. ADVANCES IN NMR SPECTROSCOPY 310

5.1 Chemical shifts 310

5.2 3[Jscr ]HNHαcoupling constants 311

5.3 Amide hydrogen exchange 312

5.4 Nuclear Overhauser Effects (NOEs) 312

6. α-HELIX – EVERYWHERE? 313

6.1 Intrinsic helix propensity equals helix content? 313

6.2 A helix propensity scale for the amino acids in TFE 314

6.3 Capping motifs and stop signals 315

6.4 Limits and population of helices as seen by CD and NMR 316

7. TURNS 317

8. β-HAIRPINS AND SHEETS 317

9. ‘CLUSTERS’ OF SIDECHAINS 320

10. THE TFE DENATURED STATE OF β-LACTOGLOBULIN 321

11. THE TFE DENATURED STATE OF HEN LYSOZYME 324

12. TERTIARY STRUCTURE, DISULPHIDES, DYNAMICS AND COMPACTNESS 327

13. PROSPECTS FOR STRUCTURE CALCULATION 328

14. EFFECT OF TFE ON QUATERNARY STRUCTURE 329

15. EFFECT ON TFE ON UN- AND REFOLDING KINETICS 330

16. OTHER USES 336

16.1 Mimicking membranes and protein receptors 336

16.2 Solubilizing peptides and proteins 336

16.3 Cosolvents as helpers for protein folding? 338

16.4 Modifying protein dynamics and catalysis 338

16.5 Effects on nucleic acids 339

16.6 Effects on lipid bilayers and micelles 339

16.7 Future applications 339

17. CONCLUSIONS: TFE – WHAT IS IT GOOD FOR? 340

18. ACKNOWLEDGMENTS 340

19. REFERENCES 340

The motivation for this review arises from the conviction that, as a result of the mass of experimental data and observations collected in recent years, the study of the physical properties of membranes is now entering a new stage of development. More and more, experiments are being designed to answer specific, detailed questions about membranes which will lead to a quantitative understanding of the way in which the physical properties of membranes are related to and influence their biological function.

1. Introduction 326

1.1 What? Terminology and general properties 327

1.2 Why? Reasons for biophysical study 329

1.3 How? Special issues for study of connexin channels 330

2. Molecular and structural context 331

2.1 Biochemical features 331

2.2 Structures 334

2.2.1 Junctional channels 335

2.2.2 Hemichannels 338

2.2.3 Heteromeric channels 342

2.2.4 Junctional plaques 347

3. Experimental approaches and issues specific to study of connexin channel physiology 349

3.1 Macroscopic currents 349

3.1.1 Junctional channels 349

3.1.2 Hemichannels 354

3.2 Single-channel currents 355

3.2.1 Junctional channels 355

3.2.2 Hemichannels 358

3.3 Molecular permeability 361

3.3.1 A selection of tracers 361

3.3.2 Junctional channels 362

3.3.3 Hemichannels 366

3.4 Other 367

4. Structural issues 368

4.1 What lines the pore? 368

4.2 Docking between hemichannels 373

4.2.1 Structural and molecular basis 374

4.2.2 Determinants of specificity of interaction 380

5. Permeability and selectivity 381

5.1 Among the usual ions 383

5.1.1 Unitary conductance 383

5.1.2 Selectivity 384

5.1.3 Nonlinear single-channel I–V relations and their molecular determinants 386

5.2 Among large permeants 391

5.2.1 Uncharged molecules 392

5.2.2 Charged molecules 393

5.2.3 Cytoplasmic/signaling molecules 396

6. Voltage sensitivity 399

6.1 Macroscopic transjunctional voltage sensitivity 404

6.2 Microscopic voltage sensitivity – Vj-gating 407

6.2.1 Molecular basis – voltage sensor 407

6.2.2 Molecular basis – transduction and/or state stability 409

6.3 Microscopic voltage sensitivity – loop gating 412

6.4 Vm-gating 414

7. Direct chemical modulation 415

7.1 Phosphorylation 417

7.2 Cytoplasmic pH and aminosulfonates 419

7.3 Calcium ion 424

7.4 Lipophiles 424

7.4.1 Long chain n-alkyl alcohols 425

7.4.2 Fatty acids and fatty acid amides 426

7.4.3 Halothane 426

7.5 Glycyrrhetinic acid and derivatives 427

7.6 Cyclic nucleotides 428

7.7 Other candidates 430

8. Connexinopathies 431

9. Summary 435

10. Acknowledgements 438

11. References 438

Connexins are the proteins that form the intercellular channels that compose gap junctions in vertebrates. Connexin channels mediate electrotonic coupling between cells and serve important functions as mediators of intercellular molecular signaling. Convincing demonstration of the latter function has been elusive, as have the experimental tools required for detailed functional study of the channels. Recently, substantial progress has been made on both fronts. Connexin channels are now known to be dynamic, multifunctional channels intimately involved in development, physiology and pathology, and amenable to study by state-of-the-art approaches. A host of developmental and physiological defects are caused by defects in connexin channels, and therefore in the intercellular molecular movement they mediate. The channel structure has been determined to 7·5 Å resolution within the plane of the membrane. Experimental paradigms have been developed that enable application of the tools of modern channel biophysics to study connexin channel structure–function. As a result, the biophysical mechanisms and biological functions of connexin channels now enjoy a vigorous and expanding experimental interest. This article focuses on the former, but with attention to issues likely to have biological consequences.

The inner membranes of mitochondria contain three multi-subunit enzyme complexes that act successively to transfer electrons from NADH to oxygen, which is reduced to water (Fig. I). The first enzyme in the electron transfer chain, NADH:ubiquinone oxidoreductase (or complex I), is the subject of this review. It removes electrons from NADH and passes them via a series of enzyme-bound redox centres (FMN and Fe-S clusters) to the electron acceptor ubiquinone. For each pair of electrons transferred from NADH to ubiquinone it is usually considered that four protons are removed from the matrix (see section 4.1 for further discussion of this point).

The concept that the liquid crystalline or mesomorphic condition was of importance to biological systems is a relatively old idea. Thus Bernal (1933) when discussing the different types of arrangements of molecules in liquid crystals commented ‘Such structures belong to the liquid crystal as a unit and not to its molecules which may be replaced by others without destroying them and they persist in spite of the complete fluidity of the substance. These are just the properties to be required for a degree of organization between that of the continuous substance, liquid or crystalline solid and even the simplest living cell.’ Stewart (1961) some thirty years later also stated that ‘It is this property – the combination of flow and lability with a preferred and relatively stable molecular orientation – that makes the mesomorphic (i.e. liquid crystal) phase uniquely appropriate to the structure of protoplasm and living tissue.’

1. Introduction 229

2. Structures 234

2.1 The structure of LH2 234

2.2 Natural variants of peripheral antenna complexes 242

2.3 RC–LH1 complexes 242

3. Spectroscopy 249

3.1 Steady-state spectroscopy 249

3.2 Factors which affect the position of the Qy absorption band of Bchla 249

4. Regulation of biosynthesis and assembly 257

4.1 Regulation 257

4.1.1 Oxygen 257

4.1.2 Light 258

4.1.2.1 AppA: blue-light-mediated regulation 259

4.1.2.2 Bacteriophytochromes 259

4.1.3 From the RC to the mature PSU 261

4.2 Assembly 261

4.2.1 LH1 262

4.2.2 LH2 263

5. Frenkel excitons 265

5.1 General 265

5.2 B800 267

5.3 B850 267

5.4 B850 delocalization 273

6. Energy-transfer pathways: experimental results 274

6.1 Theoretical background 274

6.2 ‘Follow the excitation energy’ 276

6.2.1 Bchla→Bchla energy transfer 277

6.2.1.1 B800→B800 277

6.2.1.2 B800→B850 278

6.2.1.3 B850→B850 279

6.2.1.4 B850→B875 280

6.2.1.5 B875→RC 280

6.2.2 Car[harr ]Bchla energy transfer 281

7. Single-molecule spectroscopy 284

7.1 Introduction to single-molecule spectroscopy 284

7.2 Single-molecule spectroscopy on LH2 285

7.2.1 Overview 285

7.2.2 B800 286

7.2.2.1 General 286

7.2.2.2 Intra- and intercomplex disorder of site energies 287

7.2.2.3 Electron-phonon coupling 289

7.2.2.4 B800→B800 energy transfer revisited 290

7.2.3 B850 293

8. Quantum mechanics and the purple bacteria LH system 298

9. Appendix 299

9.1 A crash course on quantum mechanics 299

9.2 Interacting dimers 305

10. Acknowledgements 306

11. References 307

This review describes the structures of the two major integral membrane pigment complexes, the RC–LH1 ‘core’ and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to ‘power’ the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.

Infrared spectroscopy is a useful technique for the determination of conformation and orientation of membrane-associated proteins and lipids. The technique is especially powerful for detecting conformational changes by recording spectral differences before and after perturbations in physiological solution. Polarized infrared measurements on oriented membrane samples have revealed valuable information on the orientation of chemical groupings and substructures within membrane molecules which is difficult to obtain by other methods. The application of infrared spectroscopy to the static and dynamic structure of proteins and peptides in lipid bilayers is reviewed with some emphasis on the importance of sample preparation. Limitations of the technique with regard to the absolute determination of secondary structure and orientation and new strategies for structural assignments are also discussed.

Protein molecules in solution or in protein crystals are characterized by rather well-defined structures in which α-helical regions, β-pleated sheets, etc., are the key features. Likewise, the double helix of nucleic acids has almost become the trademark of molecular biology as such. By contrast, the structural analysis of lipids has progressed at a relatively slow pace. The early X-ray diffraction studies by V. Luzzati and others firmly established the fact that the lipids in biological membranes are predominantly organized in bilayer structures (Luzzati, 1968). V. Luzzati was also the first to emphasize the liquid-like conformation of the hydrocarbon chains, similar to that of a liquid paraffin, yet with the average orientation of the chains perpendicular to the lipid–water interface. This liquid–crystalline bilayer is generally observed in lipid–water systems at sufficiently high temperature and water content, as well as in intact biological membranes under physiological conditions (Luzzati & Husson, 1962; Luzzati, 1968; Tardieu, Luzzati & Reman, 1973; Engelman, 1971; Shipley, 1973). In combination with thermodynamic and other spectroscopic observations these investigations culminated in the formulation of the fluid mosaic model of biological membranes (cf. Singer, 1971). However, within the limits of this model the exact nature of lipid conformation and dynamics was immaterial, the lipids were simply pictured as circles with two squiggly lines representing the polar head group and the fatty acyl chains, respectively. No attempt was made to incorporate the well-established chemical structure into this picture. Similarly, membrane proteins were visualized as smooth rotational ellipsoids disregarding the possibility that protruding amino acid side-chains and irregularities of the backbone folding may create a rather rugged protein surface.