In general the characterization of a virus particle depends upon a number of known and independently acquired parameters, all of which require a great deal of time. As a direct method of measurement, the mass of viruses has been largely ignored in virus characterization studies. Where the molecular weight of viruses has been determined by sedimentation and diffusion, sedimentation equilibrium, light scattering, or the electron microscopy-counting procedure, the relationship between this property and mass has been arrived at by employing Avogadro's number. In the present report it will be demonstrated that the disadvantages found in these usual methods of determining molecular weight, such as experimentation, calculation of virus size, and purification of virus preparation, are not present in the quantitative electron microscopy method for determination of mass and molecular weight.

Small nucleolar and Cajal body ribonucleoprotein particles (RNPs) are required for the maturation of ribosomes and spliceosomes. They consist of small nucleolar RNA or Cajal body RNA combined with partner proteins and represent the most complex RNA modification enzymes. Recent advances in structure and function studies have revealed detailed information regarding ribonucleoprotein assembly and substrate binding. These enzymes form intertwined RNA–protein assemblies that facilitate reversible binding of the large ribosomal RNA or small nuclear RNA. These revelations explain the specificity among the components in enzyme assembly and substrate modification. The multiple conformations of individual components and those of complete RNPs suggest a dynamic assembly process and justify the requirement of many assembly factors in vivo.

Nucleotide excision repair (NER) is a highly versatile and efficient DNA repair process, which is responsible for the removal of a large number of structurally diverse DNA lesions. Its extreme broad substrate specificity ranges from DNA damages formed upon exposure to ultraviolet radiation to numerous bulky DNA adducts induced by mutagenic environmental chemicals and cytotoxic drugs used in chemotherapy. Defective NER leads to serious diseases, such as xeroderma pigmentosum (XP). Eight XP complementation groups are known of which seven (XPA–XPG) are caused by mutations in genes involved in the NER process. The eighth gene, XPV, codes for the DNA polymerase ɳ, which replicates through DNA lesions in a process called translesion synthesis (TLS). Over the past decade, detailed structural information of these DNA repair proteins involved in eukaryotic NER and TLS have emerged. These structures allow us now to understand the molecular mechanism of the NER and TLS processes in quite some detail and we have begun to understand the broad substrate specificity of NER. In this review, we aim to highlight recent advances in the process of damage recognition and repair as well as damage tolerance by the XP proteins.

All organisms need homologous recombination (HR) to repair DNA double-strand breaks. Defects in recombination are linked to genetic instability and to elevated risks in developing cancers. The central catalyst of HR is a nucleoprotein filament, consisting of recombinase proteins (human RAD51 or bacterial RecA) bound around single-stranded DNA. Over the last two decades, single-molecule techniques have provided substantial new insights into the dynamics of homologous recombination. Here, we survey important recent developments in this field of research and provide an outlook on future developments.

We have discovered a well-defined extended conformation of double-stranded DNA, which we call Σ-DNA, using laser-tweezers force-spectroscopy experiments. At a transition force corresponding to free energy change ΔG = 1·57 ± 0·12 kcal (mol base pair)−1 60 or 122 base-pair long synthetic GC-rich sequences, when pulled by the 3′−3′ strands, undergo a sharp transition to the 1·52 ± 0·04 times longer Σ-DNA. Intriguingly, the same degree of extension is also found in DNA complexes with recombinase proteins, such as bacterial RecA and eukaryotic Rad51. Despite vital importance to all biological organisms for survival, genome maintenance and evolution, the recombination reaction is not yet understood at atomic level. We here propose that the structural distortion represented by Σ-DNA, which is thus physically inherent to the nucleic acid, is related to how recombination proteins mediate recognition of sequence homology and execute strand exchange. Our hypothesis is that a homogeneously stretched DNA undergoes a ‘disproportionation’ into an inhomogeneous Σ-form consisting of triplets of locally B-like perpendicularly stacked bases. This structure may ensure improved fidelity of base-pair recognition and promote rejection in case of mismatch during homologous recombination reaction. Because a triplet is the length of a gene codon, we speculate that the structural physics of nucleic acids may have biased the evolution of recombinase proteins to exploit triplet base stacks and also the genetic code.

Rhodopsins are intrinsic membrane retinal-containing proteins composed of 7 hydrophobic a-helical transmembrane columns and hydrophilic sequences of various length connecting the helices and localized at N- and C-ends of the polypeptide. The chromophore (retinal) forms a Schiff base with a lysine residue in the middle part of the last a-helix. Absorption of a photon results in isomerization of retinal which gives rise to a conformational change in the protein moiety.

Structural analyses of protein–protein interactions are required to reveal their functional mechanisms, and accurate protein–protein complex models, based on experimental results, are the starting points for drug development. In addition, structural information about proteins under physiologically relevant conditions is crucially important for understanding biological events. However, for proteins such as those embedded in lipid bilayers and transiently complexed with their effectors under physiological conditions, structural analyses by conventional methods are generally difficult, due to their large molecular weights and inhomogeneity. We have developed the cross-saturation (CS) method, which is an nuclear magnetic resonance measurement technique for the precise identification of the interfaces of protein–protein complexes. In addition, we have developed an extended version of the CS method, termed transferred cross-saturation (TCS), which enables the identification of the residues of protein ligands in close proximity to huge (>150 kDa) and heterogeneous complexes under fast exchange conditions (>0.1 s−1). Here, we discuss the outline, basic theory, and practical considerations of the CS and TCS methods. In addition, we will review the recent progress in the construction of models of protein–protein complexes, based on CS and TCS experiments, and applications of TCS to in situ analyses of biologically and medically important proteins in physiologically relevant states.

1. Introduction 373

2. Are rafts probable? 374

3. Micro-, nano- or ephemeral domains? 375

4. How can we reliably assess ‘raft’ composition? 376

5. Are rafts plausible? 379

6. What more can model systems contribute to ‘raft’ studies? 381

7. References 382

The concept of ‘lipid rafts’ and related liquid-ordered membrane microdomains has attracted great attention in the field of membrane biology, both as a novel paradigm in models of membrane organization and for the potential importance of such domains in phenomena such as membrane signaling and the differential trafficking of various membrane components. Studies of biological and of model membranes have gone hand in hand in shaping our current picture of the possible organization and functions of liquid-ordered lipid microdomains in membranes. This essay discusses some important current questions concerning the existence and functional importance of lipid microdomains in mammalian cell membranes, and the potential as well as the limitations of using model systems to help to address such questions.

DNA supercoiling is a consequence of the double-stranded nature of DNA. When a linear DNA molecule is ligated into a covalently closed circle, the two strands become intertwined like the links of a chain, and will remain so unless one of the strands is broken. The number of times one strand is linked with the other is described by a fundamental property of DNA supercoiling, the linking number (Lk).

The kink-turn (k-turn) is a widespread structural motif found in functional RNA species. It typically comprises a three-nucleotide bulge followed by tandem trans sugar edge-Hoogsteen G:A base pairs. It introduces a sharp kink into the axis of duplex RNA, juxtaposing the minor grooves. Cross-strand H-bonds form at the interface, accepted by the conserved adenine nucleobases of the G:A basepairs. Alternative acceptors for one of these divides the k-turns into two conformational classes N3 and N1. The base pair that follows the G:A pairs (3b:3n) determines which conformation is adopted by a given k-turn. k-turns often mediate tertiary contacts in folded RNA species and frequently bind proteins. Common k-turn binding proteins include members of the L7Ae family, such as the human 15·5k protein. A recognition helix within these proteins binds in the widened major groove on the outside of the k-turn, that makes specific H-bonds with the conserved guanine nucleobases of the G:A pairs. L7Ae binds with extremely high affinity, and single-molecule data are consistent with folding by conformational selection. The standard, simple k-turn can be elaborated in a variety of ways, that include the complex k-turns and the k-junctions. In free solution in the absence of added metal ions or protein k-turns do not adopt the tightly-kinked conformation. They undergo folding by the binding of proteins, by the formation of tertiary contacts, and some (but not all) will fold on the addition of metal ions. Whether or not folding occurs in the presence of metal ions depends on local sequence, including the 3b:3n position, and the −1b:−1n position (5′ to the bulge). In most cases −1b:−1n = C:G, so that the 3b:3n position is critical since it determines both folding properties and conformation. In general, the selection of these sequence matches a given k-turn to its biological requirements. The k-turn structure is now very well understood, to the point at which they can be used as a building block for the formation of RNA nano-objects, including triangles and squares.

The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.

Drug design means many things to many people. Commercially the aim is the development of compounds that can be patented and meet a variety of regulatory standards. In drug design, for medical purposes, toxicity and bio-availability are major considerations.

The understanding of flexibility and deformability in proteins is one of the current major challenges of structural molecular biology. The knowledge of the average atomic positions of three-dimensional folding of proteins, which is obtained either by X-ray diffraction or n.m.r. spectroscopy, is generally not sufficient to explain their functional mechanisms. Very often it is necessary to consider the existence of other concerted atomic motions as, for example, in the well-known case of the CO molecule fixation at the active site of myoglobin which requires the concerted displacement of a large number of atoms in order to open a channel down to this site. This opening, which depends on the physico-chemical conditions, plays the role of a regulator in the biochemical reactions (Janin & Wodak, 1983; Tainer et al. 1984; Westhof et al. 1984; Ormos et al. 1988).

The biological energy cycle of our planet is driven by photosynthesis whereby sunlight is absorbed by chlorophyll and other accessory pigments. The excitation energy is then efficiently transferred to a reaction centre where charge separation occurs in a few picoseconds. In the case of photosystem II (PSII), the energy of the charge transfer state is used to split water into oxygen and reducing equivalents. This is accomplished by the relatively low energy content of four photons of visible light. PSII is a large multi-subunit membrane protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Four high energy electrons, together with four protons (4H+), are used to reduce plastoquinone (PQ), the terminal electron acceptor of PSII, to plastoquinol (PQH2). PQH2 passes its reducing equivalents to an electron transfer chain which feeds into photosystem I (PSI) where they gain additional reducing potential from a second light reaction which is necessary to drive CO2 reduction. The catalytic centre of PSII consists of a cluster of four Mn ions and a Ca2+ linked by oxo bonds. In addition, there are seven amino acid ligands. In this Article, I discuss the structure of this metal cluster, its stability and the probability that an acid-base (nucleophilic-electrophilic) mechanism catalyses the water splitting reaction on the surface of the metal-cluster. Evidence for this mechanism is presented from studies on water splitting catalysts consisting of organo-complexes of ruthenium and manganese and also by comparison with the enzymology of carbon monoxide dehydrogenase (CODH). Finally the relevance of our understanding of PSII is discussed in terms of artificial photosynthesis with emphasis on inorganic water splitting catalysts as oxygen generating photoelectrodes.

It appears questionable whether the thermodynamics of irreversible processes can be applied formally in a simple way to the transport of matter or energy across very thin membranes such as lipid bilayers. This is because a thin layer probably cannot be described in a strict thermodynamical sense as a continuous phase.

It has virtually become a truism that osmosis, and the inseparable array of related processes comprising ultrafiltration, diffusion, anomalous osmosis, etc., has found its final and lasting exposition to which anything can only be added by way of a few finishing touches. This view, as I shall attempt to show, is quite wrong, if for no other reason than that several of the different approaches to the problem are not complementary. We want two things: to gain an insight into the mechanisms operative in porous systems, and to derive equations which predict the flow rates from a minimal set of properties – if we can decide what these are.

Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed.

The explosion of the use of paramagnetic probes to study biological problems by nuclear magnetic resonance (n.m.r.) stems from the large and easily quantifiable perturbations they produce. The main reason for these perturbations is that the magnetic moments of unpaired electrons are about 103 times greater than nuclear magnetic moments and consequently they generate much greater local fields.Such fields can give rise to large shifts in nuclear resonances; but if these fields fluctuate at the appropriate frequencies they will also cause very efficient relaxation in the nuclear resonances. It is possible to select probes that result mainly in one of these parameters being perturbed so that the terms shift and relaxation probe are now well established in the literature.

As of November 1977, the American Chemical Society's Chemical Abstracts Service computer registry of chemicals contained 4039907 distinct entities, and has been growing at an average rate of about 6000 per week. How many of these chemicals are in everyday use? Current estimates from the Environmental Protection Agency indicate that there may be as many as 50000 commonly used chemicals, not including pesticides, pharmaceuticals, and food additives. The EPA estimates that there may be as many as 1500 different active ingredients in pesticides. The Food and Drug Administration estimates that there are about 4000 active ingredients in drugs and about 2000 other ancillary compounds used in the drug industry. The FDA also estimates that there are about 2500 additives used for nutritional value and flavouring and 3000 chemicals used to preserve processed food. Thus there seem to be about 63000 chemicals in common use.

Copper (Cu) is an essential transition metal providing activity to key enzymes in the human body. To regulate the levels and avoid toxicity, cells have developed elaborate systems for loading these enzymes with Cu. Most Cu-dependent enzymes obtain the metal from the membrane-bound Cu pumps ATP7A/B in the Golgi network. ATP7A/B receives Cu from the cytoplasmic Cu chaperone Atox1 that acts as the cytoplasmic shuttle between the cell membrane Cu importer, Ctr1 and ATP7A/B. Biological, genetic and structural efforts have provided a tremendous amount of information for how the proteins in this pathway work. Nonetheless, basic mechanistic-biophysical questions (such as how and where ATP7A/B receives Cu, how ATP7A/B conformational changes and domain–domain interactions facilitate Cu movement through the membrane, and, finally, how target polypeptides are loaded with Cu in the Golgi) remain elusive. In this perspective, unresolved inquiries regarding ATP7A/B mechanism will be highlighted. The answers are important from a fundamental view, since mechanistic aspects may be common to other metal transport systems, and for medical purposes, since many diseases appear related to Cu transport dysregulation.