Many basic events which concern management and manipulation of genes within a cell – for instance transcription, replication and recombination – rely on specific interactions between proteins and nucleic acids. Such interactions are also essential for many house-keeping functions, like packing and unpacking of DNA in chromatin and assembly of ribosomes. Moreover, the details of protein–nucleic acid interplay is essential for understanding the action of viruses. The list of functional mechanisms in biology that rely on protein–DNA and protein–RNA interactions can be made much longer, but these examples represent some of the topics which motivated structural biologists to study complexes between proteins and nucleic acids as a first step beyond structure determinations of individual biomolecules.

1. INTRODUCTION 358

1.1 Summary 358

1.2 Overview 359

1.3 Four classes of pore-forming K+channel subunits – necessary and (sometimes) sufficient 361

1.4 Soluble and peripheral membrane proteins that interact with P loop subunits to alter function 362

1.5 Integral membrane proteins that interact with P loop subunits to alter function 363

2. MinK DETERMINES THE FUNCTION OF MIXED CHANNEL COMPLEXES 363

2.1 The KCNE1 gene product (MinK) gives rise to K+-selective currents and controversy 363

2.2 MinK assembles with a P loop protein, KvLQT1, to form K+channels with unique function 364

2.2.1 Single-channel conductance of KvLQT1 and MinK/KvLQT1 channels 366

2.2.2 Other differences between KvLQT1 and MinK/KvLQT1 channels 367

2.3 MinK assembles with HERG, another P loop subunit, to regulate channel activity 368

2.4 MinK does not form chloride-selective ion channels 368

3. EXPERIMENTAL AND NATURAL MinK MUTATIONS 369

3.1 Site-directed mutations 369

3.1.1 MinK mutation alters basic channel attributes and identifies key residues 369

3.1.2 MinK is a Type I transmembrane peptide 370

3.1.3 MinK is intimately associated with the IKspore 370

3.1.4 The number of MinK subunits in IKschannel complexes 372

3.2 KCNE1 mutations associated with arrhythmia and deafness alter IKschannel function 373

3.3 Summary of MinK sites critical to IKschannel function 374

4. MinK-RELATED PEPTIDES: AN EMERGING SUPERFAMILY 374

4.1 KCNE2, 3 and 4 encode MinK-related peptides 1, 2 and 3 (MiRPs) 374

4.2 MiRP1 assembles with a P loop protein, HERG, to form K+channels with unique function 375

4.2.1 MiRP1 alters activation, deactivation and single-channel conductance 376

4.2.2 MiRP1 alters regulation by K+ion and confers biphasic kinetics to channel blockade 378

4.2.3 Stable association of MiRP1 and HERG subunits 380

4.3 KCNE2 mutations are associated with arrhythmia and decreased K+flux 383

4.4 Summary of the evidence that cardiac IKrchannels are MiRP1/HERG complexes 385

5. MinK-RELATED PEPTIDES: COMMONALTIES AND IMPLICATIONS 386

5.1 Genetics and structure 386

5.2 Cell biology and function 387

6. ANSWERS, SOME OUTSTANDING ISSUES, CONCLUSIONS 387

7. ACKNOWLEDGEMENTS 389

8. REFERENCES 389

MinK and MinK-related peptide 1 (MiRP1) are integral membrane peptides with a single transmembrane span. These peptides are active only when co-assembled with pore-forming K+ channel subunits and yet their role in normal ion channel behaviour is obligatory. In the resultant complex the peptides establish key functional attributes: gating kinetics, single-channel conductance, ion selectivity, regulation and pharmacology. Co-assembly is required to reconstitute channel behaviours like those observed in native cells. Thus, MinK/KvLQT1 and MiRP1/HERG complexes reproduce the cardiac currents called IKs and IKr, respectively. Inherited mutations in KCNE1 (encoding MinK) and KCNE2 (encoding MiRP1) are associated with lethal cardiac arrhythmias. How these mutations change ion channel behaviour has shed light on peptide structure and function. Recently, KCNE3 and KCNE4 were isolated. In this review, we consider what is known and what remains controversial about this emerging superfamily.

1. INTRODUCTION 399

2. DEFINITIONS 401

3. CIS BASEPAIRS 410

3.1 Cis Watson–Crick/Watson–Crick 410

3.2 Wobble pairings 411

3.3 Cis Watson–Crick/Hoogsteen pairings 416

3.4 Bifurcated pairings 417

3.5 Cis open and water-inserted 421

4. TRANS BASEPAIRS 423

4.1 Trans Watson–Crick/Watson–Crick 423

4.2 Trans wobble pairs 424

4.3 Trans Watson–Crick/Hoogsteen pairs 424

4.4 Trans Hoogsteen/Hoogsteen pairs 430

4.5 Trans bifurcated pairings 432

5. SHALLOW-GROOVE PAIRINGS 432

5.1 Hoogsteen/Shallow-groove pairs 433

5.2 Watson–Crick/Shallow-groove pairings 438

5.3 Shallow-groove/Shallow-groove pairings 440

6. SIDE-BY-SIDE BASES 446

7. DEFINING A LIBRARY OF ISOSTERIC PAIRINGS 446

8. CONCLUSIONS 451

9. ACKNOWLEDGEMENTS 452

10. REFERENCES 452

RNA molecules fold into a bewildering variety of complex 3D structures. Almost every new RNA structure obtained at high resolution reveals new, unanticipated structural motifs, which we are rarely able to predict at the current stage of our theoretical understanding. Even at the most basic level of specific RNA interactions – base-to-base pairing – new interactions continue to be uncovered as new structures appear. Compilations of possible non-canonical base-pairing geometries have been presented in previous reviews and monographs (Saenger, 1984; Tinoco, 1993). In these compilations, the guiding principle applied was the optimization of hydrogen-bonding. All possible pairs with two standard H-bonds were presented and these were organized according to symmetry or base type. However, many of the features of RNA base-pairing interactions that have been revealed by high-resolution crystallographic analysis could not have been anticipated and, therefore were not incorporated into these compilations. These will be described and classified in the present review. A recently presented approach for inferring basepair geometry from patterns of sequence variation (Gautheret & Gutell, 1997) relied on the 1984 compilation of basepairs (Saenger, 1984), and was extended to include all possible single H-bond combinations not subject to steric clashes. Another recent review may be consulted for a discussion of the NMR spectroscopy and thermodynamic effects of non-canonical (‘mismatched’) RNA basepairs on duplex stability (Limmer, 1997).

Ion channels, like many other proteins, have moving parts that perform useful functions. The channel proteins contain an aqueous, ion-selective pore that crosses the plasma membrane, and they use a number of distinct ‘gating’ mechanisms to open and close this pore in response to biological stimuli such as the binding of a ligand or a change in the transmembrane voltage.

This review is written at a watershed in our understanding of ion channels.

1. INTRODUCTION 240

1.1 Basic structure of voltage-activated channels 241

1.2 What are the physical motions of the channel protein during gating? 243

1.3 Gating involves several distinct mechanisms of activation and inactivation 246

2. ACTIVATION GATING 246

2.1 Early evidence for an activation gate at the intracellular mouth 247

2.1.1 Open channel blockade 247

2.1.2 The ‘ foot-in-the-door’ effect 249

2.1.3 Trapping of blockers behind closed activation gates 249

2.2 Site-directed mutagenesis and the difficulty of inferring structural roles from functional effects 250

2.3 State-dependent cysteine modification as a reporter of position and motion 251

2.4 Localization of activation gating 254

2.4.1 The trapping cavity 254

2.4.2 The activation gate 255

2.4.3 Is there more than one site of activation gating? 258

3. INACTIVATION GATING 259

3.1 Ball-and-chain (N-type) inactivation 261

3.1.1 Nature of the ‘ball’ – a tethered blocking particle 262

3.1.2 The ball receptor 263

3.1.3 The chain 264

3.1.4 Variations on the N-type inactivation theme: multiple balls, foreign balls, anti-balls 265

3.2 C-type inactivation 266

3.2.1 C-type inactivation and the outer mouth of the K+channel 266

3.2.2 The selectivity filter participates in C-type inactivation 267

3.2.3 A consistent structural picture of C-type inactivation 269

3.3 By what mechanism do other voltage-gated channels inactivate? 272

4. THE VOLTAGE SENSOR 273

4.1 Quantitative principles of voltage-dependent gating 276

4.2 S4 (and its neighbours) as the principal voltage sensor 277

4.2.1 Mutational effects on voltage-dependence and charge movement 277

4.2.2 Evidence for the translocation of S4 279

4.2.3 Real-time monitoring of S4motion by fluorescence 282

4.3 Coupling between the voltage sensor and gating 283

5. CONCLUSION 284

6. ACKNOWLEDGEMENTS 287

7. REFERENCES 287

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 discovery of the double helix structure of DNA led immediately to questions on the mechanics of unravelling its intertwined strands during replication. If a parental DNA is to be duplicated into two progeny molecules by separating its two strands and copying each, then the strands must untwine rapidly during replication (Watson & Crick, 1953).

That DNA indeed replicates in such a semiconservative fashion was soon demonstrated by the Meselson–Stahl experiment (1958). At first, it appeared that the unravelling of the intertwined strands should not pose an insurmountable mechanical problem. The two strands at one end of a linear DNA, for example, can be pulled apart with concomitant rotation of the double-stranded portion of the molecule around its helical axis. If the strands of a DNA double helix are to separate at an estimated replication rate of 100000 base pairs (bp) per minute, then the speed of this rotation would be 10000 revolutions per minute from the 10 bp per turn helical geometry of the double helix. This speed, though impressive, seemed reasonable: owing to the slender rod-like shape of the double helix, the estimated viscous drag for this rotational motion is actually rather modest (Meselson, 1972).

The relationship between amino acid sequence, three-dimensional structure and biological function of proteins is one of the most intensely pursued areas of molecular biology and biochemistry. In this context, the three-dimensional structure has a pivotal role, its knowledge being essential to understand the physical, chemical and biological properties of a protein (Branden & Tooze, 1991; Creighton, 1993). Until 1984 structural information at atomic resolution could only be determined by X-ray diffraction techniques with protein single crystals (Drenth, 1994). The introduction of nuclear magnetic resonance (NMR) spectroscopy (Abragam, 1961) as a technique for protein structure determination (Wüthrich, 1986) has made it possible to obtain structures with comparable accuracy also in a solution environment that is much closer to the natural situation in a living being than the single crystals required for protein crystallography.

The term ‘amyloid’ was used originally to describe certain deposits found post- mortem in organs and tissues, which gave a positive reaction when stained with iodine (Virchow, 1854). Only later was it realized that the material was in fact predominantly proteinaceous, although it is known to be associated with carbohydrates, particularly glucosoaminoglycans, when obtained from many ex vivo sources. With the increasing precision in the definition of amyloid, initially from its characteristic green birefringence when stained with the dye Congo Red (Missmahl & Hartwig, 1953), and later from its particular appearance under the electron microscope (Cohen & Calkins, 1959) and its X-ray diffraction pattern (Eanes & Glenner, 1968), it has become evident that it is a specific fibrillar protein state, which can also be formed by some proteins when denatured in vitro (Burke & Rougvie, 1972), and by synthetic oligopeptides (Bradbury et al. 1960) that may form amyloid spontaneously when placed in pure aqueous medium (Serpell, 1996). Although these latter may form useful experimental systems for the study of amyloid, its major interest at present is that it is associated with a number of prominent lethal diseases (Benson & Wallace, 1989; Pepys, 1994).

The European Federation of Biotechnology defines biotechnology as ‘the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services’. Biotechnology thus focuses on the industrial exploitation of biological systems and is based on their unique expertise in specific molecular recognition and catalysis. The enormous potential for drug synthesis, design of biomedical diagnostics, large-scale production of biochemicals including fuels, food production, degradation of resistant wastes and extraction of raw materials will very likely make biotechnology, along with electronics and material sciences, one of the key technologies of the 21st century. From the chemical engineer's point of view, the living system participating in a biotechnological process is the central unit that catalyses chemical reactions. It exhibits a complex dependence on the bioprocess parameters, and the engineer focuses on these parameters to achieve optimal control (Hamer, 1985; Bailey & Ollis, 1986). For the natural scientist, the living system itself is in the centre of interest, so that attempts to optimize a bioprocess aim at its appropriate redesign by genetic manipulations. The increase in penicillin production by strain improvement based on random mutagenesis, which was pursued from 1940 to the mid 1970s, represents an early contribution of life scientists to improve a bioprocess that is of utmost medical importance (Hardy & Oliver, 1985).

Most advances in biology can usually be traced back to the development of a new technique: the recent explosion in sequence information in the databases arose from the pioneering work on separation methods by Frederick Sanger which paved the way for the development of protein (Sanger, 1945) and DNA/RNA (Maxam & Gilbert, 1977; Sanger, 1981) sequencing and culminated in the receipt of two Nobel prizes by Sanger. The initial phase of sequence database expansion was slow due to the tedious and slow nature of protein sequencing. Peptide sequencing was carried out manually and the complete analysis of a protein was tiresome, requiring the isolation of sufficient peptides from several digests of the target protein using proteases of different specialities to collect an overlapping set of fragments which cover the whole sequence. Protein sequencing gained momentum when the phenylisothiocyanate sequencing chemistry developed by Edman in 1949 was automated (Edman & Begg, 1967) and a commercial instrument requiring lower amounts (nanomoles) of sample was put on the market. Further technical advances such as novel valves to deal with small volumes of aggressive chemicals, the introduction of high pressure liquid chromatography (HPLC), and novel supports for sample immobilization, were all combined in the first gas phase sequencers, greatly increasing the sensitivity and allowing automated data collection (Hewick et al. 1981) and analysis. The new instruments with a sensitivity in the low picomole range appeared as rapid advances in DNA technology such as the development of restriction mapping (Danna et al. 1973), cloning (Cohen et al. 1973) and the dideoxynucleotide sequencing chemistry were threatening to make protein chemistry a relic of the past (Malcolm, 1978).

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.

The mechanism of energy transduction in biological membranes is a fascinating unsolved problem in biology. It has been recognized for some time that the driving force for a variety of seemingly unrelated phenomena (e.g. secondary active transport, oxidative phosphorylation, rotation of the bacterial flagellar motor) is a bulk-phase, transmembrane electrochemical ion gradient. However, insight into the molecular mechanisms by which free energy stored in such gradients is transduced into work or into chemical energy is just beginning. Nonetheless, gene sequencing and analyses of deduced amino-acid sequences suggest that many biological machines involved in energy transduction, secondary transport proteins in particular (Henderson, 1990; Marger & Saier, 1993), fall into families encompassing proteins from archaea to the mammalian central nervous system, thereby raising the possibility that the members may have common basic structural features and mechanisms. In addition, many of these proteins play important roles in human disease (e.g. diabetes mellitus, glucose/galactose malabsorption, some forms of drug abuse, stroke, antibiotic resistance), as well as the mechanism of action of certain psychotropic drugs.

The focus of this review is on recent observations with a specific secondary transport protein, the lactose permease (lac permease) of Escherichia coli, as a representative of a huge number of proteins that catalyse similar reactions in virtually all biological membranes.

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.

1. INTRODUCTION 195

2. THE PROTEIN SYNTHESIS SYSTEM 199

2.1 Aminoacyl-tRNA synthetases 199

2.1.1 Glutaminyl-tRNA synthetase 202

2.1.2 Glutamyl-tRNA synthetase 206

2.1.3 Tyrosyl- and tryptophanyl-tRNA synthetases 207

2.1.4 Methionyl-tRNA synthetase 208

2.1.5 Aspartyl-tRNA synthetase 208

2.1.6 Lysyl-tRNA synthetase 209

2.1.7 Seryl-tRNA synthetase 210

2.1.8 Glycyl- and histidyl-tRNA synthetases 210

2.1.9 Phenylalanyl-tRNA synthetase 211

2.2 Ribosomal proteins 211

2.2.1 L7/L12 212

2.2.2 L30 214

2.2.3 S5 215

2.2.4 S17 215

2.2.5 L6 215

2.2.6 L9 216

2.2.7 S6 217

2.2.8 L1 217

2.2.9 L14 217

2.2.10 S8 217

2.3 Elongation factors 218

2.3.1 EF-Tu 218

2.3.2 EF-G 219

3. SPLICEOSOMAL PROTEINS 221

3.1 U1 snRNP protein A 222

4. PROTEINS FROM RNA VIRUSES 223

4.1 Viral enzymes and regulatory proteins 224

4.1.1 Reverse transcriptase 224

4.1.2 Tat 224

4.1.3 Rev 225

4.2 Viral capsid proteins 225

4.2.1 Tobacco Mosaic Virus 226

4.2.2 Satellite Tobacco Mosaic Virus 226

4.2.3 Bean-Pod Mottled Virus 226

4.2.4 Black Beetle Virus and Flock House Virus 228

4.2.5 Bacteriophage MS2 228

5. OTHER RNA-BINDING PROTEINS 230

5.1 tRNA-guanine transglycosylase 230

5.2 Major cold-shock protein 230

5.3 Rop 231

5.4 Ricin 231

6. CONCLUSION 232

7. ACKNOWLEDGEMENTS 233

(I) Aims and function of the Union

(1) The objects of the International Union for Pure and Applied Biophysics are advancement of education in theScience of Biophysics. In furtherance of this aim the Council may exercise the following powers:

(a) to organize international cooperation in Biophysics and to promote communication between the variousbranches of Biophysics and allied subjects;

to encourage within each adhering body cooperation between the societies that represent the interests ofBiophysics;

to contribute to the advancement of Biophysics in all its aspects.

(2) For these purposes it shall have power:

(a) to set up commissions or bodies for special purposes;

(b) to organize international meetings and conferences;

(c) to collaborate with other scientific organizations;

(d) to act in all ways as a constituent union of the International Council of Scientific Unions in accordance with thestatutes of that body;

(e) to develop any lawful activity deemed helpful to the forwarding of its declared objects.

1. INTRODUCTION 107

2. ORGANIZATION 108

3. THE MAIN GOALS 108

4. SCIENTIFIC ACTIVITIES 109

5. CONNECTION AND COOPERATION WITH OTHER ORGANIZATIONS 109

6. PUBLICATIONS 110

7. CLOSING REMARKS 110

8. HISTORICAL TABLES 111

9. APPENDIX: IUPAB STATUTES (as modified in Amsterdam, 1996) 115

The IUPAB was founded on 2 August 1961 in Stockholm as the International Organization of Pure and Applied Biophysics. The main goal of the Organization was to promote and organize international cooperation in biophysics and molecular biology and to encourage its development and practical application all over the world. Some detailed information on the foundation of the Union was published by A. K. Solomon (first Secretary General) in Quarterly Review of Biophysics.Another summary of the activity of IUPAB was prepared by R. D. Keynes(second Secretary General) in 1976.

Most working proteins, including metabolic enzymes, transcription regulators, and membrane receptors, transporters, and ion channels, share the property of allosteric coupling. The term ‘allosteric’ means that these proteins mediate indirect interactions between sites that are physically separated on the protein. In the example of ligand-gated ion channels, the binding of a suitable ligand elicits local conformational changes at the binding site, which are coupled to further conformational changes in regions distant from the binding site. The physical motions finally arrive at the site of biological activity: the ion-permeating pore. The conformational changes that lead from the ligand binding to the actual opening of the pore comprise ‘gating’. In 1956, del Castillo and Katz suggested that the competition between different ligands at nicotinic acetylcholine receptors (nAChRs) could be explained by formation of an intermediate, ligand-bound, yet inactive state of the receptor, which separates the active state of the receptor from the initial binding of the ligand (del Castillo & Katz, 1957). This ‘binding-then-gating’, two-step model went beyond the then-prevailing drug-receptor model that assumes a single bimolecular binding reaction, and paralleled Stephenson's conceptual dichotomy of ‘affinity’ and ‘efficacy’ (Stephenson, 1956). In 1965 Monod, Wyman and Changeux presented a simple allosteric model (the MWC model) (Monod et al. 1965) that explained the cooperative binding of oxygen to haemoglobin; it was adopted as an important paradigm for ligand-gated channels soon after its initial formulation (Changeux et al. 1967; Karlin, 1967; Colquhoun, 1973).

Whenever fundamental features of living systems and their molecular basis are reviewed, the problem of timing, of time setting or free open-end running times is only marginally on the desk of research agendas, although the finite ageing as one of the features resulting from time markers is known since long. With the discovery of cellular and most important of cellfree oscillatory processes new concepts and experimental techniques were designed to approach these questions more directly leading not only to a better understanding of timing but strongly contributed to concepts for spatial pattern generation. As given in the list of contents major items in the field of intracellular and intercellular periodic reactions are reviewed in Sections 2–7 in terms of specific properties of various systems and in Section 8 in summing important features common to all oscillatory stems in chemistry and biology. Section 9 draws attention to the problem of patterning in the mesoscopic domains of living systems, which is so basic in terms of the volume dimensions specific for the cellular and subcellular reaction compartments in biology. The last chapter sets some marks on urgent problems currently approached by the combined methods of molecular genetics, biochemistry and computer technologies.

1. INTRODUCTION AND HISTORY 122

2. PERIODIC GLYCOLYSIS: TEMPORAL AND SPATIAL OSCILLATIONS 123

2.1 Cell-free glycolysis 123

2.2 Intact cells 128

2.2.1 Yeast 128

2.2.2 Myocytes 130

2.2.3 β-Cells of the islets of pancreas 131

3. CALCIUM OSCILLATIONS 131

3.1 Temporal oscillations 131

3.2 Calcium waves 136

3.3 Physiology 138

4. MICROTUBULE OSCILLATIONS 140

5. THE MITOTIC OSCILLATOR 141

6. DICTYOSTELIUM DISCOIDEUM 144

7. CARDIOMUSCULAR NETWORK 150

8. GENERAL PROPERTIES OF DYNAMIC SPATIAL PATTERNS 152

9. MICROSCOPIC PATTERNS 156

10. PERSPECTIVES 162

11. ACKNOWLEDGEMENTS 163

12. REFERENCES 163

1. INTRODUCTION 68

1.1 Receptor patterns in the plasma membrane 68

1.2 Different types of receptor patterns 71

2. METHODS TO INVESTIGATE NON-RANDOM RECEPTOR CLUSTERING 73

2.1 Fluorescence resonance energy transfer 73

2.2 Flow cytometric energy transfer measurement 78

2.3 Fluorescence anisotropy and energy transfer 79

2.4 Photobleaching energy transfer on single cells 81

2.5 Two-dimensional mapping of receptor superstructures 82

2.6 Detecting single receptor molecules 85

2.7 Chemical identification of receptor clusters 86

2.8 Electron microscopy 87

2.9 Scanning force microscopy 88

3. CONFORMATIONAL STATES OF RECEPTORS 90

3.1 Multi-subunit receptor structures 90

3.2 Physical parameters influencing conformational states 91

3.3 Chemical interactions and receptor conformations 92

4. ON THE ORIGIN OF NATURALLY OCCURRING RECEPTOR CLUSTERS 93

4.1 Synthesis of receptors and their localization in the plasma membrane 93

4.2 Lipid domain structure of the plasma membrane 94

4.3 The validity of the SingerNicolson model 94

5. CONCLUSIONS 96

6. ACKNOWLEDGEMENTS 96

7. REFERENCES 97

1. INTRODUCTION 2

2. OVERALL STRUCTURE AND SUBSTRUCTURES 5

2.1 Overall structure and components 5

2.2 Bidirectional rotary motor 5

2.3 Drive shaft 8

2.4 Bushing 8

2.5 Universal joint 9

2.6 Helical propeller 9

2.7 Axial junction 10

2.8 Capping structure 11

3. ASSEMBLY PROCESS OF THE FLAGELLUM 11

3.1 Step by step assembly 11

3.2 Flagellum-specific export apparatus and the channel 12

4. UNIQUE CHARACTERISTICS OF THE FLAGELLAR MOTOR DYNAMICS 13

5. STRUCTURAL DESIGN OF FLAGELLIN FOR ASSEMBLY REGULATION AND POLYMORPHISM 14

5.1 Domain organization and terminal disorder of flagellin 15

5.2 The role of terminal disorder in filament formation and polymorphism 17

5.3 Common structural motif for regulation of self-assembly 21

6. STRUCTURAL DESIGN OF FLAGELLAR FILAMENTS FOR POLYMORPHISM 22

6.1 Polymorphic mechanism 23

6.2 Structures of the filaments deduced by electron microscopy 25

6.2.1 Overview of the electron microscopic studies 25

6.2.2 Helical image reconstruction procedure 27

6.2.3 Structural details of the filament 28

6.3 X-ray fibre diffraction studies 32

6.3.1 Overview of the X-ray studies 32

6.3.2 Orientation of liquid crystalline sols and diffraction patterns 33

6.3.3 Equatorial analysis 35

6.3.4 A preliminary map refined at 11 resolution 37

6.4 Overall chain folding of the subunit in the filament 38

6.4.1 Mapping out the terminal and central regions 38

6.4.2 The chain folding and role of each domain 42

6.5 Polymorphic nature of flagellar filament 43

6.5.1 Comparison of the L- and R-type 43

6.5.2 New helical symmetry Lt-type 46

6.5.3 Direct comparison of the Lt-type lattice to the other two 48

6.5.4 Plausible conformational changes involved in polymorphism 51

7. PERSPECTIVE 55

8. ACKNOWLEDGEMENTS 55

9. REFERENCES 55