To deepen our understanding of the principles determining the folding and functioning of globular proteins the determination of their three-dimensional structures must be supplemented with the characterization of their internal motions. Although dynamical events in proteins occur on time-scale ranging from femtoseconds to at least seconds, the physical properties of globular proteins are such that picosecond (ps) time-scale motions make a particularly important contribution to the internal fluctuations of the atoms from their mean positions.

1. Introduction 328

2. Basic concepts of neutron scattering 329

2.1 Introduction 329

2.2 Neutron-scattering functions 331

2.3 Coherent and incoherent neutron scattering. The particular role of hydrogen in incoherent scattering 332

2.4 Total elastic scattering, EISF and mean square displacement (MSD) 333

2.5 Quasielastic scattering and relaxation function 334

2.6 Inelastic scattering and density of states 335

3. Experimental aspects and instruments 335

3.1 Energy and space resolution 335

3.2 General sample aspects 335

3.3 Potential effects of D2O on dynamics 336

3.4 Experimental 2H (deuterium) labelling 336

4. Physics of protein dynamics 336

4.1 Models 336

4.2 The dynamical transition 338

4.3 Effective force constants 339

5. Dynamics of hydrated protein powders 339

5.1 First experiments on myoglobin 340

5.2 Dynamical transitions in other proteins 340

5.3 The role of hydration water 341

5.4 Influence of the solvent 344

5.5 Diffusional motions within proteins by QENS 346

5.6 Inelastic neutron scattering and vibrational spectra 347

5.7 Conclusions 351

6. Membranes 352

6.1 Lipid bilayers 353

6.2 BR and the purple membrane (PM) 353

6.2.1 The dynamical transition in the PM 353

6.2.2 QENS from oriented PM 354

6.2.3 Hydration dependence of PM motions 355

6.2.4 Local dynamics in PM studied by isotope labelling 356

6.2.5 Dynamics of different BR conformations 357

7. Protein solutions 358

7.1 From powders to solutions 358

7.2 Water dynamics and solvent dependence of the dynamical transition in proteins 359

8. Comparing neutron scattering with other techniques 359

9. Biological relevance 360

9.1 Dynamics–activity relations 360

9.2 Dynamics–stability relations (adaptation to extreme environments) 360

9.3 Protein folding 361

10. Acknowledgements 364

11. References 364

This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.

The generation of action potentials in nerve and muscle requires cell membranes with steeply voltage-dependent ionic permeabilities. The voltage-dependent, ion-selective pathways responsible for excitation have been characterized for numerous excitable tissues such as nerve axon, muscle, electric organ, algae and epithelia (Aidley, 1971; Hodgkin, 1964; Cole, 1968). The process of activating ionic pathways by some stimulus, such as a change in membrane potential, is called gating.

Calcium ion plays an essential role in many biological processes. The environment about Ca2+ may be probed by substitution of tripositive lanthanide ions, Ln3+. Ca2+ proteins fall into two broad classes: those that are inhibited by Ln3+ substitution and those that are not. It is suggested that although Ca2+ undertakes a primarily structural role in the Ln3+ non-inhibited proteins, Ca2+ may be near the active site or participate in the mechanism of action of Ln3+ inhibited proteins. Ca2+ and Ln3+ radii are similar; most Ln3+ are slightly larger than Ca2+ in complexes of the same coordination number, and substitution of Ln3+ for Ca2+ is accommodated by a slight decrease in bond distance or by an increase in coordination number. Luminescence from Tb3+ has been demonstrated to be a sensitive environmental probe of Ca2+ binding sites in proteins.

Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT3, GABAA and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT3) and some are anion selective (e.g. GABAA and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience.

In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them.

1. Introduction 270

1.1 Overview of nucleosome structure 271

2. Relative equilibrium stability (affinity) of histone–DNA interactions in nucleosomes 272

2.1 Relative affinity equals relative equilibrium stability 272

2.2 Competition assays for relative free-energy measurements 273

2.3 Technical issues in relative free-energy measurements 275

2.4 Range of affinities 278

3. Relation of nucleosome stability to nucleosome positioning 279

3.1 Translational nucleosome positioning 279

3.2 Rotational positioning 280

3.3 Unfavorable positioning 281

3.4 Experiments 281

4. Physical basis of DNA sequence preferences 282

4.1 Free-energy cost of DNA bending 283

4.2 Molecular mechanics of DNA bending and bendability 284

4.3 Bent and bendable DNA sequences 286

4.4 Parameter sets for prediction of DNA bending and bendability 288

4.5 DNA twisting 290

4.6 Energetics of nucleosomal DNA packaging 291

5. DNA sequence motifs for nucleosome packaging 292

5.1 Natural and designed nucleosomal DNAs 293

5.2 New rules and reagents from physical selection studies 294

5.3 Molecular basis of DNA sequence preferences 299

5.4 Special properties of the TA step 300

5.5 Unfavorable sequences 302

5.6 Natural genomes 303

5.7 Evolutionary approach toward an optimal sequence 305

5.8 Optimization by design 305

6. Dynamic nucleosome instability 308

6.1 Site-exposure equilibria 308

6.2 DNA sequence-dependence to site-exposure equilibria 312

6.3 Nucleosome translocation 315

6.4 Action of processive enzymes 319

7. Conclusions 319

8. Acknowledgements 320

9. References 320

The nucleosome core particle is the fundamental repeating subunit of chromatin. It consists of two molecules each of the four ‘core histone’ proteins, H2A, H2B, H3 and H4, and a 147 bp stretch of DNA. The lowest level of chromatin organization consists of a repeated array of nucleosome core particles separated by variable lengths of ‘linker DNA’. In many, but not all, cases, each core particle plus its linker DNA is associated with one molecule of a fifth ‘linker’ histone protein, H1. The complex of the core particle plus its linker DNA and H1 (when present) is called a ‘nucleosome’.

1. Introduction 110

2. The occurrence of helical junctions in nucleic acids 111

2.1 The four-way DNA junction and genetic recombination 111

2.2 Helical junctions in RNA 112

2.3 Homology and branch migration of four-way junctions 112

2.4 Forms of helical junctions available for study 113

3. The structure of the four-way DNA junction 114

3.1 The global structure of the junction 114

3.2 The stacked X-structure 114

3.3 The junction has antiparallel character 115

3.4 The stereochemistry of the four-way DNA junction 116

3.4.1 Molecular modelling 116

3.4.2 NMR studies 116

3.4.3 Crystallography 116

4. Role of metal ions in the folding of the four-way DNA junction 122

4.1 An extended structure of the four-way junction at low salt concentrations 122

4.2 Structural interconversion between the extended and stacked X-structures 122

4.3 Location of structural metal ions in the four-way junction 124

5. Conformational variation in the four-way junction 125

5.1 Formation of alternative stacking conformers and sequence-dependent bias 125

5.1.1 Demonstration of alternative stacking conformers 125

5.1.2 Simultaneous presence of both stacking conformers 126

5.1.3 Exchange between stacking conformers 128

5.1.4 Longer-range sequence dependence 129

5.2 Variability in the interhelical angle 129

5.2.1 The interhelical angle 129

5.2.2 Variation, flexibility and malleability of the interhelical angle 130

5.3 Perturbation of the junction structure 130

6. Branch migration 131

6.1 Strand exchange between homologous sequences, and branch migration 131

6.2 The rate of branch migration 131

6.3 The effect of magnesium ions on branch migration rates 132

6.4 Branch migration and the structure of the DNA junction 132

7. Three-way DNA junctions 133

7.1 The perfectly basepaired three-way junction 133

7.2 The effect of unpaired bases; the bulged three-way junction 134

7.3 Two inequivalent stacking conformers 134

7.4 The stereochemistry of the bulged three-way junction 136

8. Helical junctions in RNA 136

8.1 The four-way junction in RNA 136

8.2 Some important four-way junctions in functional RNA species 137

8.2.1 The U1 snRNA junction 137

8.2.2 The four-way junction of the hairpin ribozyme 138

8.3 Three-way helical junctions in RNA 138

9. Recognition and distortion of four-way DNA junctions by proteins 139

9.1 Junction-resolving enzymes 139

9.1.1 Occurrence of junction-resolving enzymes 140

9.1.2 Cleavage of DNA junctions by resolving enzymes 140

9.1.3 Structure-selective binding of resolving enzymes to four-way junctions 143

9.1.4 Distortion of the structure of junctions by resolving enzymes 143

9.1.5 Relationship between distortion and cleavage of DNA junctions 144

9.2 Branch migration proteins 145

9.3 Site-specific recombinases 146

9.4 Other proteins 149

10. Summary and conclusions 149

11. Acknowledgements 151

12. References 151

Helical junctions in nucleic acids are important in biology. In DNA, their main significance is as intermediates in both homologous and site-specific recombination events. In RNA they are important architectural elements.

Helical junctions may be defined as branchpoints where double-helical segments intersect with axial discontinuities, such that strands are exchanged between the different helical sections. Thus the integrity of junctions is maintained by the covalent continuity of the component strands. Junctions can be perfectly basepaired, such that every base is paired with its Watson–Crick complement, or they can contain mismatches or unpaired bases; the latter can have significant effects on the folding of the structures. A systematic nomenclature exists for the unambiguous description of different junctions (Lilley et al. 1995) and some examples are illustrated in Fig. 1.

The purpose of the present article is to review what is known about the structures of helical junctions, and their recognition by proteins. The recent presentation of crystal structures of four-way junctions (Nowakowski et al. 1999; Ortiz-Lombardía et al. 1999; Eichman et al. 2000) provides a good opportunity to examine the current state of knowledge. We can also ask whether there are general principles behind the folding of branched nucleic acid species. Two possible principles emerge.

The success of equilibrium thermodynamics in describing static phenomena has inspired many attempts to develop a rigorous thermodynamics of rate processes.

The use of heteronuclear filters enables the editing of complex 1H nuclear magnetic resonance (NMR) spectra into simplified subspectra containing a lesser number of resonance lines, which are then more easily amenable to detailed spectral analysis. This editing is based on the creation of heteronuclear two-spin or multiple-spin coherence and discrimination between protons that do or do not participate in these heteronuclear coherences. In principle, heteronuclear editing can be used in conjunction with one-dimensional or multidimensional 1H-NMR experiments for studies of a wide variety of low-molecular-weight compounds or macromolecular systems, and is implicitely applied in a wide range of heteronuclear NMR experiments with proton detection (e.g. Bax et al. 1983; Griffey & Redfield, 1987). In the present article we shall focus on the use of heteronuclear filters in two-dimensional (2D) [1H, 1H]-NMR experiments. The selection of the material covered was primarily motivated by its impact on the practice of protein structure determination in solution, and on NMR studies of intermolecular interactions with biological macromolecules. Section 2 surveys potential applications of heteronuclear filters in this area. The remainder of the article is devoted to an introduction of the theoretical principles used in heteronuclear filters, and to a detailed description of the experimental implementation of these measurements. In writing the review we tried to minimize redundancy with the recent article in Quarterly Review of Biophysics by Griffey & Redfield (1987) and to concentrate on experiments that were introduced during the period 1986–9.

‘Tunnelling’ is the metaphorical name given to the process, possible in quantum mechanics, but not in classical mechanics, whereby a particle can disappear from one side of a potential-energy barrier and appear on the other side without having enough kinetic energy to mount the barrier. One can think of this as a manifestation of the wave-nature of particles. The wavelength is larger if a particle is lighter. In particular electrons, being very light compared to atoms, have wavelengths as large or larger than atoms at energies found in the valence shells of molecules. Thus, they easily ooze through and around atoms and molecules. We are also concerned with the tunnelling of heavy particles: nuclei, atoms, molecules.

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).

In recent decades, in silico absorption, distribution, metabolism, excretion (ADME), and toxicity (T) modelling as a tool for rational drug design has received considerable attention from pharmaceutical scientists, and various ADME/T-related prediction models have been reported. The high-throughput and low-cost nature of these models permits a more streamlined drug development process in which the identification of hits or their structural optimization can be guided based on a parallel investigation of bioavailability and safety, along with activity. However, the effectiveness of these tools is highly dependent on their capacity to cope with needs at different stages, e.g. their use in candidate selection has been limited due to their lack of the required predictability. For some events or endpoints involving more complex mechanisms, the current in silico approaches still need further improvement. In this review, we will briefly introduce the development of in silico models for some physicochemical parameters, ADME properties and toxicity evaluation, with an emphasis on the modelling approaches thereof, their application in drug discovery, and the potential merits or deficiencies of these models. Finally, the outlook for future ADME/T modelling based on big data analysis and systems sciences will be discussed.

Technology which makes use of biological materials has advanced dramatically in the last few decades. Production of specific biochemicals by selected microbial strains, the use of enzymes for stereospecific biosynthesis of materials and gene technological production of biologically important macromolecules are a few examples of these developments.

1. Introduction 362

1.1 The elusive water pores 362

1.2 CHIP28 362

2. Studies on AQP-1 363

2.1 Expression of AQP1 cDNA in Xenopus oocytes 363

2.2 Reconstitution of purified AQP1 into artificial lipid bilayers 364

2.3 Structural information deduced from the primary sequence 365

2.4 Evolution and mammalian AQPs 365

3. Chronological overview over AQP structures 368

3.1 AQP1 – the red blood cell water pore 368

3.2 GlpF – the E. coli glycerol facilitator 371

3.3 AQPZ – the E. coli water pore 372

3.4 AQP0 – the lens-specific aquaporin 373

3.5 AQP4 – the main aquaporin in brain 377

3.6 SoPiP2;1 – a plant aquaporin 379

3.7 AQPM – an archaeabacterial aquaporin 379

4. Proton exclusion 380

5. Substrate selectivity 382

6. Pore regulation 385

6.1 Hormonal regulation of AQP trafficking 385

6.2 Influence of pH on AQP water conduction 386

6.3 Regulation of AQP pore conductance by protein binding 387

6.4 Pore closure by conformational changes in the AQP0 pore 388

7. Unresolved questions 390

8. Acknowledgments 390

9. References 391

The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family.

Electrostatic effects play an essential role in specific molecular recognition and molecular assembly in many biologically important molecules. The specific electric field at the active site also regulates the catalytic reaction of a protein. Moreover, intramolecular or inter-subunit electrostatic interactions, such as saltbridges, hydrogen bonds, and charge-dipole interactions, are considered to work to stabilize protein molecules. Those electrostatic roles recently observed in proteins are reviewed with a description of the origins and principles of electrostatic forces, and analyses will be made using simple models to illuminate the physical basis.

Surfactants are surface-active, amphiphilic compounds that are water-soluble in the micro- to millimolar range, and self-assemble to form micelles or other aggregates above a critical concentration. This definition comprises synthetic detergents as well as amphiphilic peptides and lipopeptides, bile salts and many other compounds. This paper reviews the biophysics of the interactions of surfactants with membranes of insoluble, naturally occurring lipids. It discusses structural, thermodynamic and kinetic aspects of membrane–water partitioning, changes in membrane properties induced by surfactants, membrane solubilisation to micelles and other phases formed by lipid–surfactant systems. Each section defines and derives key parameters, mentions experimental methods for their measurement and compiles and discusses published data. Additionally, a brief overview is given of surfactant-like effects in biological systems, technical applications of surfactants that involve membrane interactions, and surfactant-based protocols to study biological membranes.

It has become standard practice to compare new amino-acid and nucleotide sequences with existing ones in the rapidly growing sequence databases. This has led to the recurring identification of certain sequence patterns, usually corresponding to less than 300 amino-acids in length. Many of these identifiable sequence regions have been shown to fold up to form a ‘domain’ structure; they are often called protein ‘modules’ (see definitions below). Proteins that contain such modules are widely distributed in biology, but they are particularly common in extracellular proteins.

The use of fluorescent nucleic acid base analogues is becoming increasingly important in the fields of biology, biochemistry and biophysical chemistry as well as in the field of DNA nanotechnology. The advantage of being able to incorporate a fluorescent probe molecule close to the site of examination in the nucleic acid-containing system of interest with merely a minimal perturbation to the natural structure makes fluorescent base analogues highly attractive. In recent years, there has been a growing interest in developing novel candidates in this group of fluorophores for utilization in various investigations. This review describes the different classes of fluorophores that can be used for studying nucleic acid-containing systems, with an emphasis on choosing the right kind of probe for the system under investigation. It describes the characteristics of the large group of base analogues that has an emission that is sensitive to the surrounding microenvironment and gives examples of investigations in which this group of molecules has been used so far. Furthermore, the characterization and use of fluorescent base analogues that are virtually insensitive to changes in their microenvironment are described in detail. This group of base analogues can be used in several fluorescence investigations of nucleic acids, especially in fluorescence anisotropy and fluorescence resonance energy transfer (FRET) measurements. Finally, the development and characterization of the first nucleic base analogue FRET pair, tCO–tCnitro, and its possible future uses are discussed.

1. Introduction 98

2. Historical background of two-photon effects 99

2.1 2PE 100

2.2 Harmonic generation 100

2.3 Fluorescence correlation spectroscopy 100

3. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 101

3.1 General considerations 101

3.2 Fluorescence intensity under the 2PE condition 103

3.3 Optical consequences of 2PE 104

3.4 Saturation effects in 2PE 108

3.5 Fluorescence correlation spectroscopy 109

3.5.1 Autocorrelation analysis 110

3.5.2 Photon-counting histogram analysis 112

4. Two-photon-excited probes 115

5. Design considerations for a 2PE fluorescence microscope 119

5.1 General aspects 119

5.2 Descanned and non-descanned 2PE imaging 121

5.3 Lens objectives and pulse broadening 122

5.4 Laser sources 125

5.5 Example of a practical realization 127

6. Applications 134

6.1 Biological applications of 2PE 134

6.1.1 Brain images 134

6.1.2 Applications on the kidney 139

6.1.3 Mammalian embryos 139

6.1.4 Applications to immuno-response 141

6.1.5 Myocytes 141

6.1.6 Retina 142

6.1.7 DNA imaging 143

6.1.8 FISH applications 144

6.2 2PE imaging of single molecules 144

6.3 FCS applications 148

6.4 Signals from nonlinear interactions 151

7. Conclusions 153

8. Acknowledgements 154

9. References 155

This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.