The dynamics of proteins in solution includes a variety of processes, such as backbone and side-chain fluctuations, interdomain motions, as well as global rotational and translational (i.e. center of mass) diffusion. Since protein dynamics is related to protein function and essential transport processes, a detailed mechanistic understanding and monitoring of protein dynamics in solution is highly desirable. The hierarchical character of protein dynamics requires experimental tools addressing a broad range of time- and length scales. We discuss how different techniques contribute to a comprehensive picture of protein dynamics, and focus in particular on results from neutron spectroscopy. We outline the underlying principles and review available instrumentation as well as related analysis frameworks.

In the years that have passed since the publication of Wolfram Saenger's classic book on nucleic acid structure (Saenger, 1984), a considerable amount of new data has been accumulated on the range of conformations which can be adopted by DNA. Many unusual species have joined the DNA zoo, including new varieties of two, three and four stranded helices. Much has been learnt about intrinsic DNA curvature, dynamics and conformational transitions and many types of damaged or deformed DNA have been investigated. In this article, we will try to summarise this progress, pointing out the scope of the various experimental techniques used to study DNA structure, and, where possible, trying to discern the rules which govern the behaviour of this subtle macromolecule. The article is divided into six major sections which begin with a general discussion of DNA structure and then present successively, B-DNA, DNA deformations, A-DNA, Z-DNA and DNARNA hybrids. An extensive set of references is included and should serve the reader who wishes to delve into greater detai.

X-ray diffraction patterns from crystals of biological macromolecules contain sufficient information to define atomic structures, but atomic positions are inextricable without having electron-density images. Diffraction measurements provide amplitudes, but the computation of electron density also requires phases for the diffracted waves. The resonance phenomenon known as anomalous scattering offers a powerful solution to this phase problem. Exploiting scattering resonances from diverse elements, the methods of MAD (multiwavelength anomalous diffraction) and SAD (single-wavelength anomalous diffraction) now predominate for de novo determinations of atomic-level biological structures. This review describes the physical underpinnings of anomalous diffraction methods, the evolution of these methods to their current maturity, the elements, procedures and instrumentation used for effective implementation, and the realm of applications.

1. Introduction 62

2. Channel structure 63

2.1 Isoforms, primary structure and topology of Ca2+-release channels 63

2.2 Identification of ligand binding sites within the primary sequence of RyR 65

2.2.1 Calcium 66

2.2.2 Calmodulin 66

2.2.3 FK506-binding proteins 66

2.2.4 L-type Ca2+ channel 66

2.2.5 Ryanodine 67

2.3 The three-dimensional structure of the RyR channel 68

3. Channel function 70

3.1 RyR channel gating 70

3.2 Ion translocation and discrimination 71

3.2.1 Monovalent inorganic cations 71

3.2.2 Divalent inorganic cations 74

3.2.3 Organic monovalent cations 75

3.2.4 Permeant ion translocation can be blocked 75

3.3 Summary of ion handling in RyR 76

3.4 Where is the pore and what components of RyR are involved in its formation? 76

3.5 The mechanisms underlying ion translocation and discrimination in RyR 79

3.6 Does RyR employ ion–ion repulsion to attain high unitary conductance? 82

3.6.1 Conductance–activity relationships 83

3.6.2 Concentration dependence of reversal potential 83

3.6.3 The dependence of unitary conductance on mole-fraction 83

3.6.4 Effective valence of channel-blocking cations 84

3.6.5 Modelling ionic conduction 84

3.7 Factors influencing maximum conductance 85

3.8 Factors influencing ion entry 86

3.9 Theoretical design for the pore of RyR 88

4. What do we know about the structure of the conduction pathway in RyR? 88

4.1 The narrowest region of the conduction pathway – the ‘selectivity filter’ of RyR 89

4.2 The voltage drop across RyR – the length of the ‘pore’ 89

4.3 Mechanisms for ion discrimination in RyR 93

4.4 Musings on the structure of the RyR/InsP3R pore 95

5. Summary 98

6. Acknowledgements 98

7. References 98

The purpose of this article is to provide a description of the current state of our understanding of certain aspects of the relationship between the structure and function of the ryanodine receptor (RyR). RyR is an ion channel found in the membranes of the intracellular Ca2+ storage organelles, the endoplasmic reticulum (ER) and its counterpart in muscle cells the sarcoplasmic reticulum (SR), where it provides a regulated pathway for the release of stored Ca2+ during Ca2+ signalling processes such as fertilization and muscle contraction. RyR possesses both high- and low-affinity binding sites for the plant alkaloid ryanodine; however, whilst this ligand gives the channel its name and is of toxicological and pharmacological interest, physiological regulation of channel gating is mediated by the binding of cytosolic ligands (primarily Ca2+, ATP, Mg2+) and in some cases by direct coupling with a surface membrane voltage sensor.

Detailed structural investigations on living cells are problematic because existing structural methods cannot reach high resolutions on non-reproducible objects. Illumination with an ultrashort and extremely bright X-ray pulse can outrun key damage processes over a very short period. This can be exploited to extend the diffraction signal to the highest possible resolution in flash diffraction experiments. Here we present an analysis of the interaction of a very intense and very short X-ray pulse with a living cell, using a non-equilibrium population kinetics plasma code with radiation transfer. Each element in the evolving plasma is modeled by numerous states to monitor changes in the atomic populations as a function of pulse length, wavelength, and fluence. The model treats photoionization, impact ionization, Auger decay, recombination, and inverse bremsstrahlung by solving rate equations in a self-consistent manner and describes hydrodynamic expansion through the ion sound speed. The results show that subnanometer resolutions could be reached on micron-sized cells in a diffraction-limited geometry at wavelengths between 0·75 and 1·5 nm and at fluences of 1011–1012 photons μm−2 in less than 10 fs. Subnanometer resolutions could also be achieved with harder X-rays at higher fluences. We discuss experimental and computational strategies to obtain depth information about the object in flash diffraction experiments.

Drug discovery has classically targeted the active sites of enzymes or ligand-binding sites of receptors and ion channels. In an attempt to improve selectivity of drug candidates, modulation of protein–protein interfaces (PPIs) of multiprotein complexes that mediate conformation or colocation of components of cell-regulatory pathways has become a focus of interest. However, PPIs in multiprotein systems continue to pose significant challenges, as they are generally large, flat and poor in distinguishing features, making the design of small molecule antagonists a difficult task. Nevertheless, encouragement has come from the recognition that a few amino acids – so-called hotspots – may contribute the majority of interaction-free energy. The challenges posed by protein–protein interactions have led to a wellspring of creative approaches, including proteomimetics, stapled α-helical peptides and a plethora of antibody inspired molecular designs. Here, we review a more generic approach: fragment-based drug discovery. Fragments allow novel areas of chemical space to be explored more efficiently, but the initial hits have low affinity. This means that they will not normally disrupt PPIs, unless they are tethered, an approach that has been pioneered by Wells and co-workers. An alternative fragment-based approach is to stabilise the uncomplexed components of the multiprotein system in solution and employ conventional fragment-based screening. Here, we describe the current knowledge of the structures and properties of protein–protein interactions and the small molecules that can modulate them. We then describe the use of sensitive biophysical methods – nuclear magnetic resonance, X-ray crystallography, surface plasmon resonance, differential scanning fluorimetry or isothermal calorimetry – to screen and validate fragment binding. Fragment hits can subsequently be evolved into larger molecules with higher affinity and potency. These may provide new leads for drug candidates that target protein–protein interactions and have therapeutic value.

The description of the molecular processes which underlie visual excitation is the fundamental problem in understanding vision at the level of a single photoreceptor. Thus far only a general outline of photoreceptor function has emerged with little known about actual biochemical and biophysical mechanisms.

The study of the behaviour of DNA when subjected to electric fields poses several intriguing problems of fundamental physico-chemical importance. Electric field (Kerr effect) orientation of DNA in free solution as well as migration of DNA in gel electrophoresis are two well-established, but so far rather separate, research fields. Whereas the first one has been generally concerned with basic structural and dynamical properties of DNA (Charney, 1988), the second is closely related to techniques of molecular biology (for a review on DNA electrophoresis, see stellwagen 1987).

1. Introduction 286

2. Membrane protein assembly inE. coli286

2.1. Role of the SRP 287

2.2. YidC – a translocon component devoted to membrane proteins? 287

2.3. The TAT pathway 288

2.4. ‘Spontaneous’ membrane protein insertion 288

3. Membrane protein assembly in the ER 289

3.1. How TM segments exit the translocon 289

3.2. Proteins with multiple topologies 290

3.3. Stop-transfer effector sequences 291

3.4. Non-hydrophobic TM segments? 291

3.5. ‘Frustrated’ topologies 291

3.6. N-tail translocation across the ER 292

4. Membrane protein assembly in mitochondria 292

4.1. The Oxa1p pathway 292

4.2. The TIM22/54 pathway 293

5. Evolution of membrane protein topology 293

5.1. RnfA/RnfE – two homologous proteins with opposite topologies 293

5.2. YrbG – duplicating an odd number of TMs 294

6. Genome-wide analysis of membrane proteins 295

6.1. Prediction methods 295

6.2. How many membrane proteins are there? 295

6.3. The positive-inside rule 296

6.4. Dominant classes of membrane proteins 296

7. The structure of transmembrane α-helices 296

7.1. What TM helices look like 297

7.2. The ‘helical hairpin’ 297

7.3. Prolines in TM helices 297

7.4. Charged residues in TM helices: the ‘snorkel’ effect 298

7.5. The ‘aromatic belt’ 298

8. Helix–helix packing in a membrane environment 298

8.1. Lessons learnt from glycophorin A 298

8.2. Genetic screens for helix–helix interactions 299

8.3. Statistical studies 299

8.4. Membrane protein folding 299

9. Recent 3D structures 300

9.1. KcsA – the first ion channel 300

9.2. MscL – sensing lateral pressure changes 300

9.3. The cytochrome bc 1 complex 300

9.4. Fumarate reductase 301

9.5. Bacteriorhodopsin – watching a membrane protein at work 301

10. Concluding remarks 301

11. Acknowledgements 302

12. References 302

For a variety of reasons – not the least biomedical importance – integral membrane proteins are now very much in focus in many areas of molecular biology, biochemistry, biophysics, and cell biology. Our understanding of the basic processes of membrane protein assembly, folding, and structure has grown significantly in recent times, both as a result of new methodological developments, more high-resolution structure data, and the possibility to analyze membrane proteins on a genome-wide scale.

So what is new in the membrane protein field? Various aspects of membrane protein assembly and structure have been reviewed over the past few years (Cowan & Rosenbusch, 1994; Hegde & Lingappa, 1997; Lanyi, 1997; von Heijne, 1997; Bernstein, 1998); here, I will try to bring together a number of exciting recent developments. Particularly noteworthy are the discoveries related to the mechanisms of membrane protein assembly into the inner membrane of E. coli, the inner membrane of mitochondria, and the way transmembrane segments are handled by the ER translocon.

Other advances include detailed studies of the interaction between transmembrane helices and the lipid bilayer, and of helix–helix packing interactions in the membrane environment. The availability of full genomic sequences have made it possible to study membrane proteins on a genome-wide scale. Finally, a handful of new high-resolution 3D structures have appeared.

This review will deal only with helix bundle proteins, i.e. integral membrane proteins where the transmembrane segments form α-helices. For reviews on the other major class of integral membrane proteins – the β-barrel proteins – see Schirmer (1998) and Buchanan (1999). For readers who prefer a more ‘literary’ introduction to the membrane protein field, may I suggest von Heijne (1999).

This review is a partially personal account of the discovery of virus structure and its implication for virus function. Although I have endeavored to cover all aspects of structural virology and to acknowledge relevant individuals, I know that I have favored taking examples from my own experience in telling this story. I am anxious to apologize to all those who I might have unintentionally offended by omitting their work.

The first knowledge of virus structure was a result of Stanley's studies of tobacco mosaic virus (TMV) and the subsequent X-ray fiber diffraction analysis by Bernal and Fankuchen in the 1930s. At about the same time it became apparent that crystals of small RNA plant and animal viruses could diffract X-rays, demonstrating that viruses must have distinct and unique structures. More advances were made in the 1950s with the realization by Watson and Crick that viruses might have icosahedral symmetry. With the improvement of experimental and computational techniques in the 1970s, it became possible to determine the three-dimensional, near-atomic resolution structures of some small icosahedral plant and animal RNA viruses. It was a great surprise that the protecting capsids of the first virus structures to be determined had the same architecture. The capsid proteins of these viruses all had a ‘jelly-roll’ fold and, furthermore, the organization of the capsid protein in the virus were similar, suggesting a common ancestral virus from which many of today's viruses have evolved. By this time a more detailed structure of TMV had also been established, but both the architecture and capsid protein fold were quite different to that of the icosahedral viruses. The small icosahedral RNA virus structures were also informative of how and where cellular receptors, anti-viral compounds, and neutralizing antibodies bound to these viruses. However, larger lipid membrane enveloped viruses did not form sufficiently ordered crystals to obtain good X-ray diffraction. Starting in the 1990s, these enveloped viruses were studied by combining cryo-electron microscopy of the whole virus with X-ray crystallography of their protein components. These structures gave information on virus assembly, virus neutralization by antibodies, and virus fusion with and entry into the host cell. The same techniques were also employed in the study of complex bacteriophages that were too large to crystallize. Nevertheless, there still remained many pleomorphic, highly pathogenic viruses that lacked the icosahedral symmetry and homogeneity that had made the earlier structural investigations possible. Currently some of these viruses are starting to be studied by combining X-ray crystallography with cryo-electron tomography.

Exocytosis is the fundamental process by which cells communicate with each other. The events that lead up to the fusion of a vesicle loaded with chemical messenger with the cell membrane were the subject of a Nobel Prize in 2013. However, the processes occurring after the initial formation of a fusion pore are very much still in debate. The release of chemical messenger has traditionally been thought to occur through full distention of the vesicle membrane, hence assuming exocytosis to be all or none. In contrast to the all or none hypothesis, here we discuss the evidence that during exocytosis the vesicle-membrane pore opens to release only a portion of the transmitter content during exocytosis and then close again. This open and closed exocytosis is distinct from kiss-and-run exocytosis, in that it appears to be the main content released during regular exocytosis. The evidence for this partial release via open and closed exocytosis is presented considering primarily the quantitative evidence obtained with amperometry.

1. Introduction 168

1.1 Genomic annotation of metalloproteins 168

1.2 Why NMR structures? 168

1.3 Why paramagnetic metalloproteins? 169

2. General theory 170

2.1 Nuclear and electron spins 170

2.2 Hyperfine coupling 171

2.3 The effect of the hyperfine coupling on the NMR shift: the hyperfine shift 173

2.4 The effect of the hyperfine coupling on nuclear relaxation 174

2.5 Interplay between electron spin properties and features of the NMR spectra 178

3. Paramagnetism-based structural restraints 180

3.1 Contact shifts and relaxation rates as restraints 181

3.2 Locating the metal ion within the protein frame: pseudocontact shifts 184

3.3 Cross-correlation rates 186

3.4 Residual dipolar couplings 188

3.5 Interplay between different restraints 190

4. NMR without1H detection 191

4.1 The protocol for 13C-detected protonless assignment of backbone and side-chains 194

4.2 Protonless heteronuclear NMR experiments tailored to paramagnetic systems 196

5. The use of lanthanides as paramagnetic probes 198

6. The case of Cu(II) proteins 202

7. Perspectives 208

8. Acknowledgments 209

9. References 209

Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+-binding proteins and for Ca2+-binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.

1. Introduction 206

2. Quantifying the notions behind the energy landscape 206

2.1 Basic concepts of the Random Energy Model (REM) 206

2.2 Replica-symmetric partition functions and densities of states 209

2.3 The RHP phase diagram and avoided phase transitions 210

2.4 Basic concepts of the entropy of topologically constrained polymers 212

3. Beyond the Random Energy Model 219

3.1 The GREM and the glass transition in a finite RHP 222

4. Basics of configurational diffusion for RHPs and proteins 227

4.1 Kinetics on a correlated energy landscape 231

5. Thermodynamics and kinetics of protein folding 234

5.1 A protein Hamiltonian with cooperative interactions 234

5.2 Variance of native contact energies 235

5.3 Thermodynamics of protein folding 236

5.4 Free-energy surfaces and dynamics for a Hamiltonian with pair-wise interactions 240

5.5 The effects of cooperativity on folding 242

5.6 Transition-state drift 242

5.7 Phase diagram for a model protein 245

5.8 A non-Arrhenius folding-rate curve for proteins 246

6. Non-Markovian configurational diffusion and reaction coordinates in protein folding 247

6.1 An illustrative example 250

6.2 Non-Markovian rate theory and reaction surfaces 251

6.3 Application of non-Markovian rate theory to simulation data 257

7. Structural and energetic heterogeneity in the folding mechanism 259

7.1 The general strategy 261

7.2 An illustrative example 263

7.3 Free-energy functional 264

7.4 Dependence of the barrier height on mean loop length (contact order) and structural variance 268

7.5 Illustrations using lattice model proteins and functional theory 269

7.6 Connections of functional theory with experiments 271

8. Conclusions and future prospects 273

9. Acknowledgments 274

10. Appendices

A. Table of common symbols 275

B. GREM construction for the glass transition 276

C. Effect of a Q-dependent diffusion coefficient 279

D. A frequency-dependent Einstein relation 279

11. References 281

We have seen in Part I of this review that the energy landscape theory of protein folding is a statistical description of a protein's complex potential energy surface, where individual folding events are sampled from an ensemble of possible routes on the landscape. We found that the most likely global structure for the landscape of a protein can be described as that of a partially random heteropolymer with a rugged, yet funneled landscape towards the native structure. Here we develop some quantitative aspects of folding using tools from the statistical mechanics of disordered systems, polymers, and phase transitions in finite-sized systems. Throughout the text we will refer to concepts and equations developed in Part I of the review, and the reader is advised to at least survey its contents before proceeding here. Sections, figures or equations from Part I are often prefixed with I- [e.g. Section I-1.1, Fig. I-1, Eq. (I-1.1)].

The fundamental relationship between structure and function has served to guide investigations into the workings of living systems at all levels - from the whole organism to individual cells on down to individual molecules. When X-ray crystallography began to reveal the three-dimensional structures of proteins like myoglobin, lysozyme and RNase A, protein chemists were well prepared to draw inferences about functional mechanisms from the precise positioning of amino acid residues they could see. The close proximity between an amino acid side chain and a chemical group on a bound ligand strongly suggests a functional role for that side chain in binding affinity and specificity. Likewise, the nearly universal finding of large clusters of hydrophobic side chains buried in the core of proteins strongly supports a major functional role of hydrophobic interactions in protein folding and stability. Even though eminently plausible hypotheses like these, grounded in the most fundamental principles of chemistry and the logic of structure–function relationships, become widely accepted and make their way into textbooks, protein chemists have felt compelled to search for ways to test them and put them on a more quantitative basis.

A quantitative relationship between polarization properties of fluorescence light and molecular rotational diffusion was first derived by Perrin (1926). His results, which concerned spherical particles, have later been refined to the more complex rotational motion of asymmetric bodies (Memming, 1961; Chuang & Eisenthal, 1972; Ehrenberg & Rigler, 1972; Belford, Belford & Weber, 1972).

The central points of this paper can now be summarized. We consider here, for simplicity only, vanishing particle concentration. In equilibrium sedimentation equation (6) applies. The density increment is a measurable quantity. It can either be introduced into equation (6) to calculate M2, or it can be analysed by equations (7) and (8) to provide additional information on specific volumes and solute interactions.

Light scattering is determined by the analogous equation (20). The refractive index increment is also experimentally accessible and its structure (not considered here) is similar to that of the density increment. Small angle X-ray scattering is determined by equation (31) and the electron density increment which appears in this equation cannot be directly determined by experiment. Yet it can be obtained in straightforward fashion from the mass density increment, by equation (34). Similarly, in the case of neutron scattering (equation (38)), the scattering length density increment is obtained from the mass density increment by equation (40), or it may now be directly evaluated by neutron interferometry.

DNA restriction and modification enzymes are responsible for the hostspecific barriers to interstrain and interspecies transfer of genetic information that have been observed in a variety of bacterial cell types. Although the phenomenon of host specificity was initially observed in the early 1950s (Luria & Human, 1952; Bertani & Weigle, 1953), it was nearly a decade later that Arber and his colleagues accurately predicted the molecular basis of the phenomenon. Their experiments with bacteriophage λ demonstrated that a given host-specificity system imparts a specific modification to the viral DNA, and further, that restriction of DNA lacking the appropriate modification is s consquence of nucleolytic hydrolysis upon entry into the host cell (Arber & Dussoix, 1962; Dussoix & Arber, 1962; Arber, Hattman & Dussoix, 1963).

I describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N–H insertion, C–H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists’ favorite transformations with the exquisite selectivity and tunability of enzymes.

In the last few years since the early NMR structural studies of small proteins such as glucagon (Braun et al. 1983) and lac represser headpiece (Zuiderweg et al. 1984) the quality of the structure determinations have improved considerably. Of major importance has been the introduction of phase sensitive detection in the Tl dimension (States et al. 1982; Marion & Wüthrich, 1983) which has allowed for absorption presentation of 2D data with the resulting enhancement in resolution, accuracy of coupling constant measurements and accuracy of peak volume integrations. Introduction of new pulse sequences, advances in instrumentation and further developments in the structure calculation algorithms have also helped improve the quality of NMR structural analyses of proteins.

Since its introduction, pulse radiolysis has been an important technique for examining the properties of organic and inorganic radicals, and for enumerating those reactions responsible for cellular damage by ionizing radiation. Biochemists, and biophysicists outside the area of radiation biology appear, perhaps for historical reasons, to have an incomplete appreciation of the technique's potential. Protein chemists in particular, have been only dimly aware of the numerous reports of, and the significant results obtained from pulse radiolysis studies of proteins. Our purpose here is to bring some of these results together in order to emphasize the power and usefulness of pulse radiolysis experiments both for elucidating enzyme reaction mechanisms, and for gaining information on the structure of proteins in aqueous solutions. Reviews containing related, or in part the same material to be covered here have appeared previously; for example, Land (1970), Adams et al. (1972a), Shafferman & Stein (1975), Adams & Wardman (1977). This review updates these earlier works, but more importantly approaches the topic of protein pulse radiolysis with a different emphasis.