It has been known since the time of Schultze (1866) that in the vertebrate retina there are two types of photoreceptors, rods and cones, and that they serve different visual functions; rods for scotopic vision, and cones for photopic. The terminology originates from the shape of the outer segments in which the photosensitive pigment molecules are contained. The cone outer segments are conic and taper towards the tips, while the rod outer segments are typically cylindrical. Fig. 1 is a schematic diagram from Brown, Gibbons & Wald (1963) of the ultrastructure of the rod and cone outer segments of the mudpuppy, Necturus, as studied by electron microscopy. Both appear to be made up of a pile of transverse paired membranes. In cones these arise by infolding of the plasma membrane, and in rods they have probably arisen in a similar way, but each pair of membranes is sealed around the edge so as to form a closed double-membrane disc (Sjöstrand, 1961). Because of the universal lamellation within the rod and cone outer segments, it looks as if there were no appreciable intracellular space, but yet Toyoda, Nosaki & Tomita (1969), and Toyoda et al. (1970) were successful in intracellular recording from the outer segments of single rods of the nocturnal gecko and frog.

1. Introduction 29

2. Landmarks in NMR of macromolecules 32

2.1 Protein structures and methods development 32

2.1.1 Sequential assignment method 32

2.1.2 Triple-resonance experiments 34

2.1.3 Structures of large proteins 36

2.2 Protein–nucleic acid complexes 37

2.3 RNA structures 38

2.4 Membrane-bound systems 39

3. NMR spectroscopy today 40

3.1 State-of-the-art structure determination 41

3.2 New methods 44

3.2.1 Residual dipolar couplings 44

3.2.2 Direct detection of hydrogen bonds 44

3.2.3 Spin labeling 45

3.2.4 Segmental labeling 46

3.3 Protein complexes 47

3.4 Mobility studies 50

3.5 Determination of time-dependent structures 52

3.6 Drug discovery 53

4. The future of NMR 54

4.1 The ease of structure determination 54

4.2 The ease of making recombinant protein 55

4.3 Post-translationally modified proteins 55

4.4 Approaches to large and/or membrane-bound proteins 56

4.5 NMR in structural genomics 56

4.6 Synergy of NMR and crystallography in protein structure determination 56

5. Conclusion 57

6. Acknowledgements 57

7. References 57

Since the publication of the first complete solution structure of a protein in 1985 (Williamson et al. 1985), tremendous technological advances have brought nuclear magnetic resonance spectroscopy to the forefront of structural biology. Innovations in magnet design, electronics, pulse sequences, data analysis, and computational methods have combined to make NMR an extremely powerful technique for studying biological macromolecules at atomic resolution (Clore & Gronenborn, 1998). Most recently, new labeling and pulse techniques have been developed that push the fundamental line-width limit for resolution in NMR spectroscopy, making it possible to obtain high-field spectra with better resolution than ever before (Dötsch & Wagner, 1998). These methods are facilitating the study of systems of ever-increasing complexity and molecular weight.

The experimental observations described in this article indicated that a distribution of many different fluctuations is present in a globular protein. These fluctuations were characterized by observation of many natural internal probes such as the labile peptide protons and the aromatic side chains. The conditions which are necessary to get reactions of the internal probes have been discussed in detail. The structural interpretation of the data was facilitated by the development and the use of new NMR techniques which provided the identification of the resonances of all the labile peptide protons. With NOE measurements a distinction between correlated and uncorrelated exchange events was obtained. This enabled us to elucidate the exchange mechanism over a wide range of p2H and temperature and to classify different subsets of fluctuations with respect to their lifetimes. It was further demonstrated that a change of external conditions such as temperature, p2H or pressure can change the distribution of fluctuations in the protein. The mechanisms responsible for rotation of internal aromatic side chains were also found to change with temperature, and mechanistic aspects of these fluctuations were discussed.

Visual information processing in the nervous system of flies begins with a large array of photoreceptors, which transduce a light intensity pattern, and culminates in a behavioural response that depends on that pattern.

In the previous paper we have given a quantitative description of visual control of flight orientation in the fly. This description can account for fixation, tracking and some instances of spontaneous pattern preference behaviour. The phenomenological theory outlines the basic logical organization of the visual control system of the fly. It requires the neural network between the receptors and the flight muscles to perform two main computations on the visual input. One computation extracts movement information (the term r(ψ)ψ b of the phenomenological equation). The other provides position information (the term D(ψ)).

Myriad biological processes proceed through states that defy characterization by conventional atomic-resolution structural biological methods. The invisibility of these ‘dark’ states can arise from their transient nature, low equilibrium population, large molecular weight, and/or heterogeneity. Although they are invisible, these dark states underlie a range of processes, acting as encounter complexes between proteins and as intermediates in protein folding and aggregation. New methods have made these states accessible to high-resolution analysis by nuclear magnetic resonance (NMR) spectroscopy, as long as the dark state is in dynamic equilibrium with an NMR-visible species. These methods – paramagnetic NMR, relaxation dispersion, saturation transfer, lifetime line broadening, and hydrogen exchange – allow the exploration of otherwise invisible states in exchange with a visible species over a range of timescales, each taking advantage of some unique property of the dark state to amplify its effect on a particular NMR observable. In this review, we introduce these methods and explore two specific techniques – paramagnetic relaxation enhancement and dark state exchange saturation transfer – in greater detail.

It has been more than 50 years since the elucidation of the structure of double-helical DNA. Despite active research and progress in DNA biology and biochemistry, much remains to be learned in the field of DNA biophysics. Predicting the sequence-dependent curvature and flexibility of DNA is difficult. Applicability of the conventional worm-like chain polymer model of DNA has been challenged. The fundamental forces responsible for the remarkable resistance of DNA to bending and twisting remain controversial. The apparent ‘softening’ of DNA measured in vivo in the presence of kinking proteins and superhelical strain is incompletely understood. New methods and insights are being applied to these problems. This review places current work on DNA biophysics in historical context and illustrates the ongoing interplay between theory and experiment in this exciting field.

The use of isotopic substitution is a time-honoured method for simplifying the nuclear magnetic resonance spectra of biological macromolecules. For example, the biosynthetic incorporation of a heteronucleus such as 15N or 13C into a specific amino acid residue in a protein followed by direct observation of the 15N or 13C NMR spectrum could provide a means to specifically observe a given amino acid type in that protein. By observation of the chemical shift or relaxation properties as a function of pH, ligand concentration, etc. a number of important conclusions concerning the pKa values of specific residues, the affinity of the protein for various ligands, or dynamic properties of the protein can be deduced. (See Henry et al. 1986 a, b; 1987 for an elegant modern example). In such situations, direct observation of the heteronucleus is a powerful means to observe environmental changes (Niu et al. 1979) but often these measurements are not readily interpretable in terms of alterations of protein structure. Although proton-proton dipolar interactions (NOEs) typically provide the richest source of such structural information, these interactions are not monitored in most experiments which directly observe the heteronucleus.

Most advances in biology can usually be traced back to the development of a new technique: the recent explosion in sequence information in the databases arose from the pioneering work on separation methods by Frederick Sanger which paved the way for the development of protein (Sanger, 1945) and DNA/RNA (Maxam & Gilbert, 1977; Sanger, 1981) sequencing and culminated in the receipt of two Nobel prizes by Sanger. The initial phase of sequence database expansion was slow due to the tedious and slow nature of protein sequencing. Peptide sequencing was carried out manually and the complete analysis of a protein was tiresome, requiring the isolation of sufficient peptides from several digests of the target protein using proteases of different specialities to collect an overlapping set of fragments which cover the whole sequence. Protein sequencing gained momentum when the phenylisothiocyanate sequencing chemistry developed by Edman in 1949 was automated (Edman & Begg, 1967) and a commercial instrument requiring lower amounts (nanomoles) of sample was put on the market. Further technical advances such as novel valves to deal with small volumes of aggressive chemicals, the introduction of high pressure liquid chromatography (HPLC), and novel supports for sample immobilization, were all combined in the first gas phase sequencers, greatly increasing the sensitivity and allowing automated data collection (Hewick et al. 1981) and analysis. The new instruments with a sensitivity in the low picomole range appeared as rapid advances in DNA technology such as the development of restriction mapping (Danna et al. 1973), cloning (Cohen et al. 1973) and the dideoxynucleotide sequencing chemistry were threatening to make protein chemistry a relic of the past (Malcolm, 1978).

A long-standing goal of molecular biologists has been to construct DNA-binding proteins for the control of gene expression. The classical Cys2His2 (C2H2) zinc finger design is ideally suited for such purposes. Discriminating between closely related DNA sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins (ZFPs) to target genes specifically.

Zinc fingers were discovered in 1985, arising from the interpretation of our biochemical studies on the interaction of the Xenopus protein transcription factor IIIA (TFIIIA) with 5S RNA. Subsequent structural studies revealed its three-dimensional structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc (Zn) ion ligated to a pair of cysteines and a pair of histidines and also by an inner structural hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome.

The zinc finger design can be used to construct DNA-binding proteins for specific intervention in gene expression. By fusing selected zinc finger peptides to repression or activation domains, genes can be selectively switched off or on by targeting the peptide to the desired gene target. It was also suggested that by combining an appropriate zinc finger peptide with other effector or functional domains, e.g. from nucleases or integrases to form chimaeric proteins, genomes could be modified or manipulated.

The first example of the power of the method was published in 1994 when a three-finger protein was constructed to block the expression of a human oncogene transformed into a mouse cell line. The same paper also described how a reporter gene was activated by targeting an inserted 9-base pair (bp) sequence, which acts as the promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that, by combining zinc fingers with other effector or functional domains, e.g. from nucleases or integrases, to form chimaeric proteins, genomes could be manipulated or modified.

Several applications of such engineered ZFPs are described here, including some of therapeutic importance, and also their adaptation for breeding improved crop plants.

1. Introduction 100

2. Molecular architecture 107

2.1 Primary structure 108

2.1.1 Homologous regions 109

2.1.2 Chain typing 115

2.1.3 Post-translational modifications 117

2.2 Secondary structure 118

2.2.1 Central rod domain 118

2.2.2 Head and tail domains 119

2.3 Tertiary structure 123

2.3.1 Coiled-coil rod domain 123

2.3.1.1 Specificity through salt bridges 124

2.3.1.2 Specificity through apolar interactions 127

2.3.1.3 A consensus trigger sequence for two-stranded coiled-coils 128

2.3.2 Discontinuities in the rod domain 128

2.3.2.1 Links 129

2.3.2.2 Stutter 131

2.3.3 Head and tail domains 131

2.4 Electron microscope observations 133

3. Assembly 136

3.1 Role of the coiled-coil rod domain 137

3.2 Role of end domains 141

3.3 Experimentally induced crosslinks and modes of assembly 145

3.4 Naturally occurring crosslinks for tissue coordination 154

3.5 STEM data 154

4. Quaternary structure 160

4.1 Protofilaments and protofibrils 160

4.2 Head and tail domains 163

4.3 Surface lattice structure 164

4.4 Crystal studies on intermediate filament fragments 168

5. Polymorphism 169

5.1 Variations on a theme 170

5.1.1 Axial structure 170

5.1.2 Lateral structure 171

6. Keratin intermediate filament chains in diseases 172

7. Concluding remarks 175

8. Acknowledgments 176

9. References 176

Three types of intracellular filament have been identified in eukaryotic cells, and together they constitute the key elements of the cytoskeleton. They are the microtubules, the actin-containing microfilaments and the intermediate filaments. The uniqueness of the former two types of filament in cells has been well known for a long time but, in contrast, the intermediate filaments have been a relative new-comer to the scene. The microtubules and the microfilaments have always been easy to distinguish from one another on the grounds of their respective sizes (microtubules are about 25 nm in diameter and microfilaments are about 7–10 nm in diameter). Additionally, microtubules were capable of being disaggregated by the action of colchicine, and microfilaments could be disassembled by other drugs or be decorated with heavy meromyosin to generate arrowhead-like structures. Importantly, in both microtubules and microfilaments the constituent protein subunits were arranged to give the filaments a directionality, and the ability of these filaments to function in vivo depended crucially on this feature of their structure. Microtubules, for example, are involved in mitosis, motility and transport within the cell: each of these functions is clearly a ‘directional’ one. With this background the discovery and characterization of the intermediate filaments can begin.

Fluorescence has been used in biochemical studies for many years but only recently has the information content and the practical applicability of the fluorescence method been fully realized.

Following the early studies of Newton (1954) and Weber (11954) and after the initial utilization of fluorescent probes by Chance and coworkers (Azzi et al. 1969) and Tasaki et al. (1968), in the study of membranes, the use of fluorescence to provide structural information at microscopic or molecular levels in biological membranes has become widespread. widespread. The application of the fluorescence technique to biological systems has progressed parallel to the development of a theoretical basis for fluorescence data interpretation and the synthesis of a large number of fluorescent probes, organic molecules having fluorescence characteristics that are dependent on their environment.

The compound 5-bromouracil (BrU) may be incorporated into DNA in place of its analog thymine. This review is concerned with the photochemical lesions produced by the action of ultraviolet light on such BrU-DNA, and consequent biological effects of such lesions.

Single unit recordings have provided us with a basis for understanding the auditory system, especially about how it behaves under stimulation with simple sounds such as clicks and tones. The experimental as well as the theoretical approach to single unit studies has been dichotomous. One approach, the more familiar, gives a representation of nervous system activity in the form of peri-stimulus-time (PST) histograms, period histograms, iso-intensity rate curves and frequency tuning curves. This approach observes the neural output of units in the various nuclei in the auditory nervous system, and, faced with the random way in which the neurons respond to sound, proceeds by repeatedly presenting the same stimulus in order to obtain averaged results. These are the various histogram procedures (Gerstein & Kiang, 1960; Kiang et al. 1965).

The European Federation of Biotechnology defines biotechnology as ‘the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services’. Biotechnology thus focuses on the industrial exploitation of biological systems and is based on their unique expertise in specific molecular recognition and catalysis. The enormous potential for drug synthesis, design of biomedical diagnostics, large-scale production of biochemicals including fuels, food production, degradation of resistant wastes and extraction of raw materials will very likely make biotechnology, along with electronics and material sciences, one of the key technologies of the 21st century. From the chemical engineer's point of view, the living system participating in a biotechnological process is the central unit that catalyses chemical reactions. It exhibits a complex dependence on the bioprocess parameters, and the engineer focuses on these parameters to achieve optimal control (Hamer, 1985; Bailey & Ollis, 1986). For the natural scientist, the living system itself is in the centre of interest, so that attempts to optimize a bioprocess aim at its appropriate redesign by genetic manipulations. The increase in penicillin production by strain improvement based on random mutagenesis, which was pursued from 1940 to the mid 1970s, represents an early contribution of life scientists to improve a bioprocess that is of utmost medical importance (Hardy & Oliver, 1985).

RNA molecules perform a wide variety of biological functions, from enzymic activity to storage and propagation of genetic information.

A satisfactory understanding of the functions of the sodium pump, the system responsible for the active transport of sodium and potassium, require the isolation and characterization of its protein and lipid components which are integrated in the structure of the cell membrane. The enzyme system (Na+ + K+)-ATPase, is located in membrane fragments and behaves in the test tube like the transport system in the intact cell membrane (Skou,1957) Purified preparations of this enzyme will contain some, if not all, of the components of the sodium pump.

1. Introduction 3

2. Background 5

3. 2D crystals 7

4. 1D crystals (helical arrays) 8

5. Icosahedral single particles 8

6. Single particles with lower symmetry 9

7. Cellular and subcellular electron tomography 10

8. Conclusion and future prospects 10

9. References 11

Structural analysis by electron microscopy of biological macromolecules or macromolecular assemblies embedded in rapidly frozen, vitreous ice has made great advances during the last few years. Electron cryo-microscopy, or cryo-EM, can now be used to analyse the structures of molecules arranged in the form of two-dimensional crystals, helical arrays or as single particles with or without symmetry. Although it has been possible, using crystalline or helical specimens, to reach a resolution adequate to build atomic models (4 Å), there is every hope this will soon also be possible with single particles. Small and large single particles present different obstacles to progress.

A fair beginning has been made in understanding the Mössbauer spectra of many types of haem proteins. The diamagnetic compounds have all the usual difficulties associated with the calculation of any quadrupole spectra, with the added complication of larger molecules and much less crystallographic data. In the ferric paramagnets the large molecules are quite helpful in that they prevent interaction between neighbouring sites and make possible long electron spin-relaxation times. The resulting magnetic Mössbauer spectra contain a large amount of information and, by their complexity, tend to be a guard against ambiguity in interpretation. The theoretical problem of the magnetic hyperfine interaction appears more complex than the quadrupole interaction, but this apparent complexity is only superficial in the sense that it requires more complex mathematical manipulations. The underlying physical problem is much simpler in the magnetic case because it involves interactions only with the relatively few unpaired electrons, and is insensitive to all others and has no lattice contribution. Fortunately the unpaired electrons are those which lie high in energy, and are most likely to be involved in the chemical activity of the enzyme. Most of the results on ferric haems have been fairly satisfactory, and it seems likely that many of the uncertainties will be cleared up by further work. From the physicist's point of view it appears reasonable to expect that the search for better agreement between theory and paramagnetic spectra may be a route to a better understanding of the factors affecting the quadrupole interaction. The integral spin paramagnets are an intermediate case, usually requiring large applied field to reveal their magnetic properties. No high-spin ferrous haems have yet been subjected to detailed magnetic experimental or theoretical investigation, but this should be a fruitful field.

The method of choice to reveal the conformation of protein molecules in atomic detail has been X-ray single-crystal analysis. Since the first structural analysis of diffraction patterns, computer calculations have been an important tool in these studies (Blundell & Johnson, 1976). As is described by Sheldrick (1985), it has been taken for granted that a necessary first step in the determination of a protein structure would be writing computer programs to fit structure factors. In contrast the combined use of the structural analysis of NMR data and computer calculations has been quite limited. An early attempt of such structural calculations was the quantitative determination of mononucleotide conformations in solution using lanthanide ion shifts (Barry et al. 1971).

Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.