Theoretical as well as experimental studies of translational accuracy have most often been concerned with the selection of aminoacyl-tRNA by codon-programmed ribosomes. The selection of the successive codons on the mRNA has received much less attention, probably because it represents both conceptually and experimentally, a much more demanding physical problem. Nevertheless, it would seem that errors in the selection of the codon are potentially much more destructive than errors in selection of aminoacyl-tRNA species. This can be appreciated from the following.

Until recently there has been very limited information concerning the molecular geometry and conformation of biogenic monoamines and this lack of knowledge has seriously hampered efforts to unravel the structure—function relationships at the molecular level. Nevertheless, several theories have been proposed regarding interaction between the monoamines and their receptor sites. This is especially true for the neurohumoral transmitters dopamine, noradrenaline (norepinephrine) and serotonin (5-hydroxytryptamine), the target areas of which seem to be specific not only in the sense of chemical structure but also from the steric point of view. The well-known differences in biological activity due to chiralty illustrate the importance of the three-dimensional architecture of the molecules and indicate also the presence of stereospecific receptor sites. A detailed knowledge of the conformation of the biogenic monoamines would not only elucidate the requirements upon a natural or synthetic monoamine for proper function but also give indirect information about the geometry of the receptor sites.

In this review, we discuss recent advances in biophysical virology, presenting experimental and theoretical studies on the physical properties of viruses. We focus on the double-stranded (ds) DNA bacteriophages as model systems for all of the dsDNA viruses both prokaryotic and eukaryotic. Recent studies demonstrate that the DNA packaged into a viral capsid is highly pressurized, which provides a force for the first step of passive injection of viral DNA into a bacterial cell. Moreover, specific studies on capsid strength show a strong correlation between genome length, and capsid size and robustness. The implications of these newly appreciated physical properties of a viral particle with respect to the infection process are discussed.

Introduction 351

Part I. A succession of concepts 352

1. Cooperativity 352

2. Cosolvent interaction 352

3. Linearity 354

4. Solvent exchange 354

5. Excluded volume 356

6. Summation 357

Part II

1. The Kirkwood–Buff approach 357

Acknowledgments 360

References 360

In Part I the history of progress in the stabilization and destabilization of protein conformations by means of cosolvents is outlined in terms of distinct conceptual steps. In Part II it is shown that a straightforward application of the Kirkwood–Buff theory of solutions leads to formulas for the preferential interaction and the free energy of unfolding, which confirm and generalize the results of Part I.

The capacity of biological macromolecules to act as exceedingly sophisticated and highly efficient cellular machines – switches, assembly factors, pumps, or motors – is realized through their conformational transitions, that is, their folding into distinct shapes and selective binding to other molecules. Conformational transitions can be induced, monitored, and manipulated by pulling individual macromolecules apart with an applied force. Pulling experiments reveal, for a given biomolecule, the relationship between applied force and molecular extension. Distinct signatures in the force–extension relationship identify a given biomolecule and thus serve as the molecule's ‘mechanical fingerprints’. But, how can these fingerprints be decoded to uncover the energy barriers crossed by the molecule in the course of its conformational transition, as well as the associated timescales? This review summarizes a powerful class of approaches to interpreting single-molecule force spectroscopy measurements – namely, analytically tractable approaches. On the fundamental side, analytical theories have the power to reveal the unifying principles underneath the bewildering diversity of biomolecules and their behaviors. On the practical side, analytical expressions that result from these theories are particularly well suited for a direct fit to experimental data, yielding the important parameters that govern biological processes at the molecular level.

Isomorphous replacement methods have been revolutionary in macromolecular structure research. This has been most clearly demonstrated in protein crystallography. Specific binding of heavy atoms to protein molecules in the crystalline state provides the necessary reference for the phase determination of each Bragg reflexion and opens the way to the direct structure determination to atomic resolution from X-ray diffraction.

Are isomorphous replacement methods applicable to the investigation of non-crystalline structures as well? And if so, would there be any reasonable advantage?

An answer has been given by neutron scattering. Macromolecular assemblies of nucleic acids, proteins and lipids are most easily studied in H2O /D2O mixtures, as the scattering density of the solvent can be adjusted to any of the chemically different partial structures (Stuhrmann, 1974).

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).

DNA transposases are enzymes that catalyze the movement of discrete pieces of DNA from one location in the genome to another. Transposition occurs through a series of controlled DNA strand cleavage and subsequent integration reactions that are carried out by nucleoprotein complexes known as transpososomes. Transpososomes are dynamic assemblies which must undergo conformational changes that control DNA breaks and ensure that, once started, the transposition reaction goes to completion. They provide a precise architecture within which the chemical reactions involved in transposon movement occur, but adopt different conformational states as transposition progresses. Their components also vary as they must, at some stage, include target DNA and sometimes even host-encoded proteins. A very limited number of transpososome states have been crystallographically captured, and here we provide an overview of the various structures determined to date. These structures include examples of DNA transposases that catalyze transposition by a cut-and-paste mechanism using an RNaseH-like nuclease catalytic domain, those that transpose using only single-stranded DNA substrates and targets, and the retroviral integrases that carry out an integration reaction very similar to DNA transposition. Given that there are a number of common functional requirements for transposition, it is remarkable how these are satisfied by complex assemblies that are so architecturally different.

Learning and memory are important elements of our daily lives, familiar to all through introspection. Yet the mechanisms underlying these processes are still for the most part unknown. Here are problems which combine a maximum of intrinsic and practical interest with a minimum of actual knowledge and understanding. Years of our lives are dedicated to the formation of certain long-term memories and behaviour patterns, yet we have only rudimentary notions of how such ‘schooling’ is best accomplished. There is no certainty in any aspect of the process. We are not sure whether relatively few cells or millions participate in a memory trace; whether these cells change as a whole, or whether the changes are limited to synaptic regions. In fact, we cannot be certain whether the changes are confined to the neurones or whether the glia also participate.

The discovery of nucleosomes as a basic repeating unit of the chromatin structure organizing the major part of eukaryotic DNA greatly catalyzes the expansion of our knowledge on chromatin. Several lines of experimental evidence have led to the formulation of the nucleosome conception: the observation of chains of globular particles in electron micrographs of chromatin (Olins & Olins, 1974); the demonstration that DNA is released as a set of discrete sizes upon digestion of chromatin with endogenous nucleases (Hewish & Burgoyne, 1973); the isolation of discrete nucleoprotein particles upon digestion of chromatin with micrococcal nuclease (Rill & Van Holde, 1973).

Small-angle neutron scattering studies of biological macromolecules have developed rapidly in recent years due to the advent of high neutron-flux reactors,† efficient position-sensitive area detectors for neutrons (Allemand et al. 1975) and novel design of instruments (Ibel, 1976). At the same time position-sensitive detectors for X-rays (Gabriel & Dupont, 1972) have revitalized the small-angle X-ray scattering field (Kratky & Pilz, 1972; Luzzati et al. 1976) and it is now becoming clear that neutrons and X-rays can provide complementary structural information on biological complexes.

1. Introduction 430

2. Transformations of the RecA filament 431

2.1 The different forms of the RecA filament 431

2.2 Orientation and position of the RecA monomers in the active filament 433

2.3 Transmission of structural information along the filament 433

3. RecA-induced DNA deformations 435

3.1 Characteristics of RecA-bound DNA 435

3.2 Stretching properties of double-stranded DNA 436

3.3 DNA bound to architectural proteins 437

3.4 Implications for RecA-induced DNA deformations 438

3.5 Axial distribution of the DNA stretching deformation 438

4. Contacts between RecA and the DNA strands 440

4.1 The DNA-binding sites 440

4.2 Possible arrangement of loops L1 and L2 and the three bound strands of DNA 442

5. Strand arrangement during pairing reorganization 444

5.1 Hypotheses for DNA strand association 444

5.2 Association via major or minor grooves 446

5.3 Post-strand exchange geometries 446

6. Conclusion 447

7. Acknowledgments 448

8. References 448

Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

Biological cells are defined by the membrane that shields their vital molecules from the environment. The lipid bilayer of the membrane is an effective dielectric shield (Parsegian, 1969; Andersen, 1978; Honig et al. 1986), preventing penetration by charged molecules: the lipid presents a large electrostatic energy barrier because it cannot neutralize the charge of solute molecules nearly as well as water. This energy barrier inhibits the permeation of solutes with local charge, even metabolites. Of course, metabolites do enter cells, and so physiologists have suspected (for a very long time, Hille, 1984, ch. 8) that the membrane shield is pierced by aqueous channels, through which solutes diffuse (with their local charge substantially neutralized) as they cross the membrane. These aqueous pores now have molecular reality (e.g. Noda et al. 1984; Miller, 1986). Each is formed by a specialized protein, integral to membranes, perhaps shaped like a thick-walled pipe, called ionic channels. Channels control the movement of many important molecules in and out of cells by the ‘gating’ mechanism that controls their opening and closing and by the selective properties of their open channel.

To a certain degree, the events underlying the action potential are understood. For each ion to which the membrane is permeable, there exists an equilibrium potential, whose physical origin is in the activity gradient for that ion. The membrane potential is then the summation of these individual ionic batteries each weighted according to the membrane permeability to that ion. In nerve fibres at rest the potassium permeability is relatively high and the membrane potential is near to the equilibrium potential for potassium. During excitation there is a transitory increase in the permeability to sodium and a slower transitory increase in the potassium permeability also. The membrane potential, therefore, temporarily moves to a value near to the sodium equilibrium potential and then returns to its resting value. Less well understood are the molecular mechanisms responsible for these selective changes in membrane permeability, i.e. how it is that a channel (pathway bywhich an ion traverses the membrane) changes its availability for ion passage as a response to a change in the membrane potential. This is the gating process.

In earlier papers (Wyman, 1975a, b, 1981) I have called attention to the existence of a group of linkage potentials derivable from one another by a corresponding group of Legendre transformations. These potentials are applicable to any system in a state of equilibrium but are especially useful in the study of the behaviour, or, as we may say, the functional chemistry, of a macromolecule in the presence of its ligands. In this paper I show how these potentials may be represented by a set of contour graphs which embody the results of observation and are transformable into one another by a group of graphical permutations equivalent to the Legendre group.

Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington's Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several μm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 µm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.

1. Introduction 189

2. Channel properties 191

2.1 Voltage-dependent gating 191

2.2 Ion permeability 193

2.2.1 Selectivity between potassium and chloride 193

2.2.2 Permeability to large cations and large anions 193

2.3 Single-channel characteristics 194

2.4 Molecularity of the channel 195

3. Colicin Ia channel topology and protein translocation 195

3.1 Channels formed by whole colicin Ia 195

3.1.1 General channel topology 196

3.1.2 The translocated region 199

3.1.3 The nonuniqueness of the upstream membrane-inserted segment 199

3.2 Channels formed by the C-terminal domain of colicin Ia 200

4. Concluding remarks 202

5. Acknowledgement 203

6. References 203

Colicins are plasmid-encoded proteins, produced by some strains of E. coli, that kill other strains lacking the specific immunity protein encoded by the same plasmid. Most of the colicins have a three-domain structure: a central domain that binds to a receptor in the outer membrane of the target cell; an N-terminal domain that interacts with target cell proteins to move the C-terminal domain across the outer membrane and periplasmic space to the inner membrane; and a C-terminal domain that carries the toxic activity. In some colicins the C-terminal domain is an enzyme that kills the cell by entering the cytoplasm and attacking its DNA (e.g. colicin E2), its ribosomal RNA (e.g. colicin E3), or another target (Schaller et al. 1982; Ogawa et al. 1999). In other colicins, the C-terminal domain forms an ion-conducting channel in the inner membrane that ultimately leads to cell death by allowing essential solutes to leak out of the cell. These colicins, or their isolated C-terminal domains, can also form voltage-dependent channels in planar phospholipid bilayers. (For a review of the E colicins, including enzymatic colicins, see James et al. 1996; for a review of channel-forming colicins, see Cramer et al. 1995; and for a review of colicin import into E. coli, see Lazdunski et al. 1998.) The channel-forming colicins are the subject of this review, with particular emphasis on one member of this group, colicin Ia, and the protein translocation associated with the gating of its channel.

This review is concerned primarily with the physical structure and changes in the structure of RNA molecules. It will be evident that we have not attempted comprehensive coverage of what amounts to a vast literature. We have tried to stay away from particular areas that have been recently reviewed elsewhere. Citations to and information from them are included, however, so that access to the literature is available. Much of what we treat in depth deals with the crystal structures and solution behaviour of model RNA compounds, including synthetic polymers and molecular fragments such as dinucleoside phosphates. Sequence data on natural RNA are cited, but not in detail. Similarly, apart from tRNA, natural RNAs the structural determinations of which are presently not so far advanced, are not dwelt upon. We have tried to present in detail the available structural data with scaled drawings that permit facile comparisons of molecular geometries.

Gramicidin A (GA) is a linear pentadecapeptide made of alternating D and L residues, in which the N-and C-terminals are blocked by a formyl group (head) and an ethanolamine end (tail), respectively (Sarges & Witkop, 1964):

The primary energy sources for all the organisms living on the Earth are either sunlight or the energy liberated during chemical transformations (mainly, oxidation) of certain substances – food. Within the cell this energy is transformed, accumulated, and then utilized to ensure a multitude of processes (synthesis of new low- and high-molecular compounds, muscle contraction, luminescence, transfer of ions counter to their concentration gradients, etc.).

The role of universal ‘energy keeper’, of the, as it were, ‘energy small change’ in biology is played by the molecules of adenosine tri- phosphate (ATP) whose hydrolytic dissociation in water solutions with the formation of adenosine diphosphate (ADP) and inorganic phosphate (P1) is accompanied by a rather strong decrease of system energy.†