The magnetic state of iron in haem proteins has long been recognized as a convenient indicator of chemical coordination as well as more subtle biochemical properties. In the trivalent case in particular, there are three magnetically distinct configurations of the five 3d electrons, yielding spin states of S = 1/2, 3/2 and 5/2. The first and third are extremely familiar, and often lie close enough in energy so that a thermal mixture of low-spin S = 1/2 and high-spin S = 5/2 states exists in an ensemble of molecules. Selection rules for common perturbations (ΔS = ∘, ± 1 for the spin-orbit interaction and ΔS = ∘ for the electronic Zeeman interaction) ensure that quantum mixtures, in which the wave function is a true combination of S = 1/2 and S = 5/2 components, are not observed. The mid-spin, S = 3/2, state, and allowed 5/2–3/2 and 1/2–3/2 quantum mixtures including it, are much less well known. These have only rarely been invoked in recent years as an explanation for experimental haem protein magnetic data.

When light impinges on photosynthetic material – a plant leaf, an alga or a photosynthetic bacterium – it is absorbed by an array of lightcollecting pigments. Through resonant energy transfer the absorbed quantum of light is transported to a trap, the reaction centre. Within such a trap, a specialized (bacterio)chlorophyll complex is able to eject from its excited state an electron. This electron is ‘captured’ by an adjacent acceptor, which in turn donates the electron to a second acceptor, and so on. Thus, light energy is converted into chemical energy which is ultimately used in the metabolic processes of the cell.

Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational design of proteins from first-principles, or de novo, was once considered to be impossible, but today proteins with a variety of folds and functions have been realized. We review the evolution of the field from its earliest days, placing particular emphasis on how this endeavor has illuminated our understanding of the principles underlying the folding and function of natural proteins, and is informing the design of macromolecules with unprecedented structures and properties. An initial set of milestones in de novo protein design focused on the construction of sequences that folded in water and membranes to adopt folded conformations. The first proteins were designed from first-principles using very simple physical models. As computers became more powerful, the use of the rotamer approximation allowed one to discover amino acid sequences that stabilize the desired fold. As the crystallographic database of protein structures expanded in subsequent years, it became possible to construct proteins by assembling short backbone fragments that frequently recur in Nature. The second set of milestones in de novo design involves the discovery of complex functions. Proteins have been designed to bind a variety of metals, porphyrins, and other cofactors. The design of proteins that catalyze hydrolysis and oxygen-dependent reactions has progressed significantly. However, de novo design of catalysts for energetically demanding reactions, or even proteins that bind with high affinity and specificity to highly functionalized complex polar molecules remains an importnant challenge that is now being achieved. Finally, the protein design contributed significantly to our understanding of membrane protein folding and transport of ions across membranes. The area of membrane protein design, or more generally of biomimetic polymers that function in mixed or non-aqueous environments, is now becoming increasingly possible.

1. Summary 169

2. Introduction 170

3. Sanger's method and other enzymic methods 170

3.1 Random approach 171

3.2 Direct approach 171

3.3 Enzyme technology 175

3.4 Sample preparation 175

3.5 Labels and DNA labelling 176

3.5.1 Radioisotopes 176

3.5.2 Chemiluminescent detection 176

3.5.3 Fluorescent dyes 177

3.6 Fragment separation and analysis 180

3.6.1 Electrophoresis 180

3.6.2 Mass spectrometry – an alternative 182

4. Maxam & Gilbert and other chemical methods 183

5. Pyrosequencing – DNA sequencing in real time by the detection of released PPi 187

6. Single molecule sequencing with exonuclease 190

7. Conclusion 192

8. Acknowledgements 192

9. References 193

The four best known DNA sequencing techniques are reviewed. Important practical issues covered are read-length, speed, accuracy, throughput, cost, as well as the automation of sample handling and preparation. The methods reviewed are: (i) the Sanger method and its most important variants (enzymic methods); (ii) the Maxam & Gilbert method and other chemical methods; (iii) the PyrosequencingTM method – DNA sequencing in real time by the detection of released pyrophosphate (PPi); and (iv) single molecule sequencing with exonuclease (exonuclease digestion of a single molecule composed of a single strand of fluorescently labelled deoxynucleotides). Each method is briefly described, the current literature is covered, advantages, disadvantages, and the most suitable applications of each method are discussed.

Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding.

Formation of protein–ligand complexes causes various changes in both the receptor and the ligand. This review focuses on changes in pK and protonation states of ionizable groups that accompany protein–ligand binding. Physical origins of these effects are outlined, followed by a brief overview of the computational methods to predict them and the associated corrections to receptor–ligand binding affinities. Statistical prevalence, magnitude and spatial distribution of the pK and protonation state changes in protein–ligand binding are discussed in detail, based on both experimental and theoretical studies. While there is no doubt that these changes occur, they do not occur all the time; the estimated prevalence varies, both between individual complexes and by method.The changes occur not only in the immediate vicinity of the interface but also sometimes far away. When receptor–ligand binding is associated with protonation state change at particular pH, the binding becomes pH dependent: we review the interplay between sub-cellular characteristic pH andoptimum pH of receptor–ligand binding.It is pointed out that there is a tendency for protonation state changes uponbinding to be minimal at physiologically relevant pH for each complex (no net proton uptake/release), suggesting that native receptor–ligand interactions have evolved to reduce the energy cost associated with ionization changes. As a result, previously reported statistical prevalence of these changes – typically computed at the same pH for all complexes – may be higher than what may be expected at optimum pH specific to each complex. We also discuss whether proper account of protonation state changes appears to improve practical docking and scoring outcomes relevant to structure-based drug design. An overview of some of the existing challenges in the field is provided in conclusion.

The three-dimensional structures of proteins are among the most valuable contributions of biophysics to the understanding of biological systems (Dickerson & Geis, 1969; Creighton, 1983). Protein structures are utilized in the description and interpretation of a wide variety of biological phenomena, including genetic regulation, enzyme mechanisms, antibody recognition, cellular energetics, and macroscopic mechanical and structural properties of molecular assemblies. Virtually all of the information currently available about the structures of proteins at atomic resolution has been obtained from diffraction studies of single crystals of proteins (Wyckoff et al, 1985). However, recently developed NMR methods are capable of determining the structures of proteins and are now being applied to a variety of systems, including proteins in solution and other non-crystalline environments that are not amenable for X-ray diffraction studies. Solid-state NMR methods are useful for proteins that undergo limited overall reorientation by virtue of their being in the crystalline solid state or integral parts of supramolecular structures that do not reorient rapidly in solution. For reviews of applications of solid-state NMR spectroscopy to biological systems see Torchia and VanderHart (1979), Griffin (1981), Oldfield et al. (1982), Opella (1982), Torchia (1982), Gauesh (1984), Torchia (1984) and Opella (1986). This review describes how solid-state NMR can be used to obtain structural information about proteins. Methods applicable to samples with macroscopic orientation are emphasized.

The deposition of amyloid- and amyloid-like fibrils is the main pathological hallmark of numerous protein misfolding diseases including Alzheimer's disease, transmissible spongiform encephalopathy, and type 2 diabetes. Besides the well-established role in disease, recent work on a variety of organisms ranging from bacteria to humans suggests that amyloid fibrils can also convey biological functions. To better understand the molecular mechanisms by which amyloidogenic proteins misfold in disease or perform biological functions, structural information is essential. Although high-resolution structural analysis of amyloid fibrils has been challenging, a combination of biophysical approaches is beginning to unravel the various structural features of amyloid fibrils. Here we review these recent developments with particular emphasis on amyloid fibrils that have been studied using site-directed spin labeling and electron paramagnetic resonance spectroscopy. This approach has been used to define the precise location of fibril-forming core regions and identify local secondary structures within such core regions. Perhaps one of the most remarkable findings arrived at by site-directed spin labeling was that most fibrils that contain an extensive core region of ∼20 amino acids or more share a common parallel in-register arrangement of β strands. The preference for this arrangement can be explained on topological grounds and may be rationalized by the maximization of hydrophobic contact surface.

An understanding of molecular interactions is essential for insight into biological systems at the molecular scale. Among the various components of molecular interactions, electrostatics are of special importance because of their long-range nature and their influence on polar or charged molecules, including water, aqueous ions, proteins, nucleic acids, carbohydrates, and membrane lipids. In particular, robust models of electrostatic interactions are essential for understanding the solvation properties of biomolecules and the effects of solvation upon biomolecular folding, binding, enzyme catalysis, and dynamics. Electrostatics, therefore, are of central importance to understanding biomolecular structure and modeling interactions within and among biological molecules. This review discusses the solvation of biomolecules with a computational biophysics view toward describing the phenomenon. While our main focus lies on the computational aspect of the models, we provide an overview of the basic elements of biomolecular solvation (e.g. solvent structure, polarization, ion binding, and non-polar behavior) in order to provide a background to understand the different types of solvation models.

It is our purpose to review recent experiments on haemoglobin in order to discuss them in terms of the two state model of cooperativity. Excellent previous reviews are available of the chemistry of haemoglobin (Antonini & Brunori, 1971; Gibson, 1959b) which are referred to when possible. The plethora of data necessitates that a selection must be made in a review. An intentionally wide range of experiments is selected to exhibit

Immunoglobulins are somewhat unusual in that they are biologically active proteins capable of carrying out several different functions. Thus, they can bind specifically with antigen, activate the complement system, mediate many cytotropic reactions, and act as antigen receptors on lymphocyte membranes.

In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class.

Mηδέν ἄγαν—nothing in excess. These words of the old Greek saying might well find a place in modern Biology. The maintenance of order and balance—homeostasis, as the physiologist would have it—is an attribute of all living things. At a molecular level the study of control and regulation, involving the conjugate concepts of structure and function is a central theme of Biophysics. How is it that an enzyme, by the braking effect of its end products, is prevented from going too far, or, alternatively, by the accumulation of other metabolic substances in its environment, is brought into play? How is it that a working respiratory protein like haemoglobin, by the reciprocal action of its metabolic opposite, carbon dioxide, is aided in the taking up of oxygen in the lungs and the giving it off in the tissues? These are problems of linkage. From one point of view they may be thought of in terms of information— the communication between ligand and macromolecule and between one part of the macromolecule and another. From a different point of view they may be thought of in terms of the storage and transfer of energy, of the way in which the work of introducing ligand, the same or different, at another. The analysis may be pitched either at the phenomenological, or at the mechanistic, level; but whatever the approach it profits cosiderably from the introduction of the concept of the binding potential.

Nucleoside triphosphates are crucial mediators of life. The high energy phosphoanhydride bond of (usually) ATP is used to drive unfavorable chemical reactions, to fuel biological machines, and to regulate a vast number of processes via phosphorylation of proteins. GTP, in turn, is used almost exclusively for the regulation of signal transduction and transport processes, whereas the other nucleotides play a less important role, except in synthesis pathways involving sugars (UTP) and phospholipids (CTP) and as building blocks of polynucleotides such as RNA and DNA. Proteins that bind and use these nucleotides for enzymatic reaction and regulation are very diverse. Although some of them constitute the largest protein superfamilies known (e.g. protein kinases), others seem to be far less conserved in evolution.

Proteins provide much of the scaffolding for life, as well as undertaking a variety of essential catalytic reactions. These characteristic functions have led us to presuppose that proteins are in general functional only when well structured and correctly folded. As we begin to explore the repertoire of possible protein sequences inherent in the human and other genomes, two stark facts that belie this supposition become clear: firstly, the number of apparent open reading frames in the human genome is significantly smaller than appears to be necessary to code for all of the diverse proteins in higher organisms, and secondly that a significant proportion of the protein sequences that would be coded by the genome would not be expected to form stable three-dimensional (3D) structures. Clearly the genome must include coding for a multitude of alternative forms of proteins, some of which may be partly or fully disordered or incompletely structured in their functional states. At the same time as this likelihood was recognized, experimental studies also began to uncover examples of important protein molecules and domains that were incompletely structured or completely disordered in solution, yet remained perfectly functional. In the ensuing years, we have seen an explosion of experimental and genome-annotation studies that have mapped the extent of the intrinsic disorder phenomenon and explored the possible biological rationales for its widespread occurrence. Answers to the question ‘why would a particular domain need to be unstructured?’ are as varied as the systems where such domains are found. This review provides a survey of recent new directions in this field, and includes an evaluation of the role not only of intrinsically disordered proteins but also of partially structured and highly dynamic members of the disorder–order continuum.

The scientific community will remember Peter Läuger as an exceptional man combining a generous personality and a sharp and skilful mind. He was able to attract by his views the interest of a large spectrum of biologists concerned by the mechanism of ion translocation through membranes. Yet, he was not a man with a single technique or theory. Using an authentically multidisciplinary approach, his ambition was to ‘understand transmembrane transport at the microscopic level, to capture its dynamics in the course of defined physiological processes’ (1987). According to him, ‘new concepts in the molecular physics of proteins’ had to be imagined, and ‘the traditional static picture of proteins has been replaced by the notions that proteins represent dynamic structures, subjected to conformational fluctuations covering a very wide time-range’ (1987).

The basic principles underlying fluctuation phenomena in thermodynamics have long been understood (for reviews see Kubo, 1957; Kubo, Matsuo & Kazuhiro 1973 Lax, 1960). Classical examples of how fluctuation analysis can provide an insight into the corpuscular nature of matter are the determination of Avogadro's number according to Einstein's theory of Brownian motion (see, e.g. Uhlenbeck & Ornstein, 1930; Kac, 1947) and the evaluation of the electronic charge from the shot noise in vacuum tubes (see Van der Ziel, 1970).

Few fields of biochemistry have seen such widespread applications of physical theories and techniques as that of biological oxidation. There are obvious reasons for this. Oxidation-reduction reactions form the foundations of bioenergetics, an area which can only be understood in terms of thermodynamic theory. Most components of the mitochondrial respiratory chain contain transition metals, and these elements and their chemical environment can often be studied by modern spectroscopic methods, such as electron-paramagnetic resonance (EPR). The relation between spectroscopic properties and chemical structure of metallo-proteins, e.g. haem proteins, represents one of the few branches of present-day biochemistry to which quantum mechanical calculations can profitably be applied (see, for example, Zerner, Gouterman & Kobayashi, 1966).

The adsorption of proteins at the solid-liquid interface is a process of fundamental importance in nature. Extensive reviews (MacRitchie, 1978; Andrade & Hlady, 1986; Norde, 1986) testify to the strong interest which has been shown in the problem during the past few decades. Norde & Lyklema (1978) have rightly pointed out that protein adsorption is scientifically intriguing; the phenomenology is complicated and includes many presently apparently irreconcilable observations.

The viruses have been an attractive model for the study of basic mechanisms of protein/protein and protein/nucleic acid interactions involved in the assembly of macromolecular aggregates. This has been due primarily to their relative genetic simplicity as compared to their structural and functional complexity. Although most of the initial studies were carried out on bacterial and plant viruses, increasing data has also been accumulated from animal viruses, which has led to an understanding of some basic principles, as well as to many specific strategies in every system. The study of virus assembly has been a source of ideas that underlie our present knowledge of the organization of biological systems. It has also provided, since the production of bacteriophage mutants which have allowed the study of assembly intermediates, the first system in which the genetic studies played a dominant role. The increasing volume of data over the last years has revealed how the structural components can interact sequentially through an ordered pathway to yield macromolecular assemblies that satisfy the demands of stability required for a successful transfer of genetic information from host to host.