The apparent and often spectacular movements of animals and insects, movements of the whole organism in relation to its surroundings arising from internally generated forces, have always been, by their very ubiquity, uppermost in our perception of motion in the living world. Movement in plants, generally of one organ in relation to the whole plant, whilst sometimes spectacular, have often in the past been seen as rather esoteric events, amusing perhaps, but of little importance in the general biological scheme of things. However, this is not so; plant movements are quite widespread in occurrence and all are most probably manifestations of a single physiological process, the change in volume of special motor cells. One particular movement, the opening and closing of stomata, which provides a basic control of photosynthesis, is of fundamental importance to the existence of the whole biosphere.

Growing interest in gene targeting drugs has inspired the development of a multitude of nucleic acid analogues, many of which feature substitutions in the phosphodiester moiety of the backbone (reviewed by Mesmaeker et al. 1995 and Nielsen, 1995). Peptide nucleic acid (PNA) is an example of a more radical redesign of DNA. The entire sugar-phosphate backbone is substituted by a chain of peptide-like N-(2-aminoethyl)glycine units so that an achiral and uncharged DNA-mimic is obtained (Fig. 1; Nielsen et al. 1991). The synthesis is based on standard peptide chemistry (Christensen et al. 1995) and has been automated. PNA can relatively easily be modified to include modifications of the backbone as well as of the bases (Hyrup & Nielsen, 1996). PNA is chemically stable and, in contrast to natural nucleic acids and peptides, PNA is expected to remain intact in living cells since it is not a substrate for natural hydrolytic enzymes and is not degraded by cell extracts (Demidov et al. 1994).

Electron microscopy (EM) is an important tool for high-resolution structure determination in applications ranging from condensed matter to biology. Electronic detectors are now used in most applications in EM as they offer convenience and immediate feedback that is not possible with film or image plates. The earliest forms of electronic detector used routinely in transmission electron microscopy (TEM) were charge coupled devices (CCDs) and for many applications these remain perfectly adequate. There are however applications, such as the study of radiation-sensitive biological samples, where film is still used and improved detectors would be of great value. The emphasis in this review is therefore on detectors for use in such applications. Two of the most promising candidates for improved detection are: monolithic active pixel sensors (MAPS) and hybrid pixel detectors (of which Medipix2 was chosen for this study). From the studies described in this review, a back-thinned MAPS detector appears well suited to replace film in for the study of radiation-sensitive samples at 300 keV, while Medipix2 is suited to use at lower energies and especially in situations with very low count rates.

The performance of a detector depends on the energy of electrons to be recorded, which in turn is dependent on the application it is being used for; results are described for a wide range of electron energies ranging from 40 to 300 keV. The basic properties of detectors are discussed in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE) as a function of spatial frequency.

Photosynthesis is the conversion of the quantum energy of light into the chemical energy of complex organic molecules and organized cellular structures in plants and in some bacteria. The processes of photosynthesis span the time domain of subpicoseconds to the millennia of slow-growing trees, its study brings together such diverse disciplines as photophysics, biochemistry, botany and ecology. In the last few decades tremendous progress has been made in understanding the multivarious chemical reactions that ultimately lead to the fixation of carbon dioxide into organic substance, yet the basic mechanism underlying the conversion of photon energy into chemical energy still remains very much an enigma. These so-called primary reactions which transduce the excitation energy of excited chlorophyll pigments into the potential energy of stabilized, separated charges on electron donor and electron acceptor molecules have been studied with a variety of physical techniques, among which fast optical spectroscopy and electron paramagnetic resonance (EPR) are prominent. This review will highlight one intriguing aspect of EPR, namely that of electron spin polarization (ESP).† It will be shown that ESP of photosynthetic primary reactants offers a unique tool to gain insight in the electrostatic and magnetic interactions that make photosynthesis work. Moreover, it will become apparent that ESP in photosynthesis has several unique traits not (yet) found in ESP of photochemical reactions in vitro. As such, it may serve as a paradigma of ESP phenomena and will present an absorbing spectacle also for EPR spectroscopists outside photosynthesis.

Following the first determinations of protein structures in the late 1950s and the early 1960s (see for example Kendrew et al. 1960; Perutz, 1964), the three-dimensional structures of several hundred proteins have been elucidated by X-ray diffraction on single crystals. By the end of 1991, approximately 150 entries of proteins with substantially different sequences and a well resolved structure (Hobohm et al. 1992) were deposited in the Protein Data Bank (Bernstein et al. 1977; Abola et al. 1987). In addition, many structures of homologous proteins or of mutants have been described, bringing the total number of entries to about 600. While it was soon accepted that almost all of these structures do indeed give a correct picture of the fold of the active protein in spite of the non-physiological environment of single crystals, it is not clear to what extent structural details are reliably described by these structures. In particular the surface of a protein may be modified due to the dense packing of protein molecules in the crystal lattice. A detailed knowledge of the protein surface is, however, essential for the understanding of the function of the protein.

The complex molecular events responsible for coordinating chromosome replication and segregation with cell division and growth are collectively known as the cell cycle. Progression through the cell cycle is orchestrated by the interplay between controlled protein synthesis and degradation and protein phosphorylation. Protein degradation is primarily regulated through the ubiquitin proteasome system, mediated by two related E3 protein ubiquitin ligases, the Skp1 cullin F-box (SCF) and the anaphase promoting complex (also known as the cyclosome) (APC/C). The APC/C is a multi-subunit cullin-RING E3 ubiquitin ligase that regulates progression through the mitotic phase of the cell cycle and controls entry into S phase by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D-box and KEN-box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. The aim of this article is to discuss the APC/C from a structural and mechanistic perspective. Although an atomic structure of the APC/C is still lacking, a combination of genetic, biochemical, electron microscopy studies of intact APC/C and crystallographic analysis of individual subunits, together with analogies to evolutionarily related E3 ligases of the RING family, has provided deep insights into the molecular mechanisms of catalysis and substrate recognition, and structural organisation of the APC/C.

Cytochromes are found in all biological oxidation Systems which involve transport of reducing equivalents through organized chains of membrane bound intermediates, regardless of the ultimate oxidant (Keilin, 1966; Bartsch, 1978; Meyer & Kamen, 1982). Thus, cytochromes are present not only in the aerobic mitochondrial and bac-terial respiratory chain, but are also found in much more diversified procariotic Systems, including all varieties of facultative anaerobes (nitrate and nitrite reducers), obligate anaerobes (sulphate reducers and phototrophic sulphur bacteria), facultative photoheterotrophes (phototrophic non-sulphur purple bacteria), and the photoautotrophic cyanobacteria (blue-green algae). Among the different types of cytochromes occurring in the cell, the soluble c-type cytochromes (‘class I’, Meyer & Kamen, 1982) are the most abundant and best characterized group of proteins (Bartsch, 1978; Meyer & Kamen, 1982; Dickerson & Timkovitch, 1975; Lemberg & Barrett, 1973; Salemme, 1977; Ferguson-Miller, Brautigan & Margoliash, 1979). The amino acid sequences of more than 80 mitochrondrial and close to 40 bacterial cytochromes c are known (Meyer & Kamen, 1982; Dickerson & Timkovitch, 1975; Schwartz & Dayhoff, 1976; Dayhoff & Barker, 1978).

The principle of the microprobe analysis of chemical elements is illustrated in Fig. i. Some kind of radiation is directed on to the specimen, generating signals characteristic of the elements present. Local analysis in situ is achieved in one of two ways. Most often the impinging beam is finely focused so that the signal at any moment comes only from a selected microregion. Alternatively, in some instruments, the impinging beam floods a larger region but the emergent signals characteristic of a particular element may be selected and focused to give an elemental ‘map’ or ‘image’.

In all scattering phenomena the scattering angles are inversely related to the dimensions of the scattering particles. As macromolecules are giant compared with the normally used CuKα wavelength λ = 1·54 Å, we find the scattering of the particle as a whole at Correspondingly small angles.

1. Introduction 326

2. Instrumentation 328

2.1 Instruments to study mechanical properties of RNA 328

2.1.1 AFM 328

2.1.2 Magnetic tweezers 328

2.1.3 Optical tweezers 330

2.2 Optical trap instrumentation 330

2.3 Calibrations 332

2.3.1 Calibration of trap stiffness 332

2.3.2 Calibration of force 333

2.3.3 Calibration of distance 334

2.4 Types of experiments 334

2.4.1 Force-ramp 334

2.4.2 Force-clamp or constant-force experiments 335

2.4.3 Extension-clamp or constant extension experiments 335

2.4.4 Force-jump, Force-drop 336

2.4.5 Passive mode 336

3. Thermodynamics 336

3.1 Reversibility 336

3.2 Gibbs free energy 337

3.2.1 Stretching free energy 338

3.2.1.1 Rigid molecules 338

3.2.1.2 Compliant or flexible molecules 339

3.2.2 Free energy of a reversible unfolding transition 339

3.2.3 Free energy of unfolding at zero force 340

3.2.4 Free energy of an irreversible unfolding transition 340

3.2.4.1 Jarzynski's method 341

3.2.4.2 Crooks fluctuation theorem 343

4. Kinetics 345

4.1 Measuring rate constants 345

4.1.1 Hopping 345

4.1.2 Force-jump, Force-drop 347

4.1.3 Force-ramp 348

4.1.4 Instrumental effects 350

4.2 Kinetic mechanisms 351

4.2.1 Free-energy landscapes 351

4.2.2 Kinetics of unfolding 353

5. Relating force-measured data to other measurements 354

5.1 Thermodynamics 354

5.2 Kinetics 357

6. Acknowledgements 357

7. References 358

Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.

Our concern here will be with energetic rather than kinetic aspects of how a macromolecular enzyme molecule or enzyme complex in a biological membrane accomplishes the transfer or transduction of one kind of small-molecule free energy into another. An example is the use of the free energy of ATP hydrolysis (to form ADP and P1) to move Ca2+ across a membrane against its own electrochemical potential gradient (Stryer, 1981). In this case, the free energy decreases in the hydrolysis reaction but much of this is recovered in the form of an increase in free energy of the Ca2+ ions (the remainder is dissipated as heat). There is, of course, no net change in the free energy of the enzyme itself (Ca-ATPase).

In all contemporary models of enzyme catalysis, the functional activity of enzymes is directly associated with the dynamic properties of proteins (see Blumenfeld, 1977; Chernavaskaya & Chernavskii, 1977; Volkenstein, 1975; Austin et al. 1975; Shaitan & Rubin, 1985; Ansari et al. 1985). This has recently led to a much greater interest in the dynamical properties of proteins.

Fast-folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast-folding proteins has provided insight into the mechanisms, which allow some proteins to find their native conformation well <1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even ‘slow’ folding processes: fast folders are small; relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast-folding proteins and provides an overview of the major findings of fast-folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general, as well as some work that is left to do.

Theoretical methods of conformational analysis have been applied to the biopolymers with beneficial consequences for the elucidation of structure–function relationships in these molecules. The methodology of theoretical conformational analysis and the fruits of its application to the biopolymers, particularly the polypeptides, have been the subject of numerous reviews (Ramachandran & Sasisekharan, 1968; Scheraga, 1968; Venkatachalam & Ramachandran, 1969; Scheraga, 1971; Brant, 1972; Hopflnger, 1973). A critical evaluation of these techniques as applied to the polynucleotides has recently appeared (Olson, 1975 a, b).

The DNA of all organisms has a complex and essential topology. The three topological properties of naturally occurring DNA are supercoiling, catenation, and knotting. Although these properties are denned rigorously only for closed circular DNA, even linear DNA in vivo can have topological properties because it is divided into topologically separate subdomains (Drlica 1987; Roberge & Gasser, 1992). The essentiality of topological properties is demonstrated by the lethal consequence of interfering with topoisomerases, the enzymes that regulate the level of DNA supercoiling and that unlink DNA during its replication (reviewed in Wang, 1991; Bjornsti, 1991; Drlica, 1992; Ullsperger et al. 1995).

1. Introduction 374

2. Metals in biology 378

3. The targets: structure and function of ion channels 380

4. General effects of metal ions on channels 382

4.1 Three types of general effect 382

4.2 The main regulators 383

5. Effects on gating: mechanisms and models 384

5.1 Screening surface charges (Mechanism A) 387

5.1.1 The classical approach 387

5.1.1.1 Applying the Grahame equation 388

5.1.2 A one-site approach 391

5.2 Binding and electrostatically modifying the voltage sensor (Mechanism B) 391

5.2.1 The classical model 391

5.2.1.1 The classical model as state diagram – introducing basic channel kinetics 392

5.2.2 A one-site approach 395

5.2.2.1 Explaining state-dependent binding – a simple electrostatic mechanism 395

5.2.2.2 The relation between models assuming binding to smeared and to discrete charges 396

5.2.2.3 The special case of Zn2+ – no binding in the open state 396

5.2.2.4 Opposing effects of Cd2+ on hyperpolarization-activated channels 398

5.3 Binding and interacting non-electrostatically with the voltage sensor (Mechanism C) 398

5.3.1 Combining mechanical slowing of opening and closing with electrostatic modification of voltage sensor 400

5.4 Binding to the pore – a special case of one-site binding models (Mechanism D) 400

5.4.1 Voltage-dependent pore-block – adding extra gating 401

5.4.2 Coupling pore block to gating 402

5.4.2.1 The basic model again 402

5.4.2.2 A special case – Ca2+ as necessary cofactor for closing 403

5.4.2.3 Expanding the basic model – Ca2+ affecting a voltage-independent step 404

5.5 Summing up 405

6. Quantifying the action: comparing the metal ions 407

6.1 Steady-state parameters are equally shifted 407

6.2 Different metal ions cause different shifts 408

6.3 Different metal ions slow gating differently 410

6.4 Block of ion channels 412

7. Locating the sites of action 412

7.1 Fixed surface charges involved in screening 413

7.2 Binding sites 413

7.2.1 Group 2 ions 414

7.2.2 Group 12 ions 414

8. Conclusions and perspectives 415

9. Appendix 416

10. Acknowledgements 418

11. References 418

Metal ions affect ion channels either by blocking the current or by modifying the gating. In the present review we analyse the effects on the gating of voltage-gated channels. We show that the effects can be understood in terms of three main mechanisms. Mechanism A assumes screening of fixed surface charges. Mechanism B assumes binding to fixed charges and an associated electrostatic modification of the voltage sensor. Mechanism C assumes binding and an associated non-electrostatic modification of the gating. To quantify the non-electrostatic effect we introduced a slowing factor, A. A fourth mechanism (D) is binding to the pore with a consequent pore block, and could be a special case of Mechanisms B or C. A further classification considers whether the metal ion affects a single site or multiple sites. Analysing the properties of these mechanisms and the vast number of studies of metal ion effects on different voltage-gated ion channels we conclude that group 2 ions mainly affect channels by classical screening (a version of Mechanism A). The transition metals and the Zn group ions mainly bind to the channel and electrostatically modify the gating (Mechanism B), causing larger shifts of the steady-state parameters than the group 2 ions, but also different shifts of activation and deactivation curves. The lanthanides mainly bind to the channel and both electrostatically and non-electrostatically modify the gating (Mechanisms B and C). With the exception of the ether-à-go-go-like channels, most channel types show remarkably similar ion-specific sensitivities.

Around half of all protein structures solved nowadays using solution-state nuclear magnetic resonance (NMR) spectroscopy have been because of automated data analysis. The pervasiveness of computational approaches in general hides, however, a more nuanced view in which the full variety and richness of the field appears. This review is structured around a comparison of methods associated with three NMR observables: classical nuclear Overhauser effect (NOE) constraint gathering in contrast with more recent chemical shift and residual dipole coupling (RDC) based protocols. In each case, the emphasis is placed on the latest research, covering mainly the past 5 years. By describing both general concepts and representative programs, the objective is to map out a field in which – through the very profusion of approaches – it is all too easy to lose one's bearings.

The ability of an organism to cope with foreign substances (antigens) depends in part on its capacity to synthesize antibodies (immunoglobulins) of the proper binding specificity to recognize and combine with these antigens. In view of the great variety of possible antigens, antibodies, or more specifically their combining sites, display considerable variation and possess structural properties such as to enable them to bind the antigenic determinants. A vast amount of immunoglobulin sequence data has become available and the three-dimensional structures of a number of immunoglobulin fragments have been elucidated. With these results we can now begin to understand the structural aspects of antibody–antigen reactions. The crystallographic results and the sequence data have been reviewed elsewhere (Capra & Kehoe, 1975; Davies, Padlan & Segal 1975a, b; Gally, 1973; Kabat, 1976; Nisonoff, Hopper & Spring, 1975; Poljak, 1975a, b). Here, an attempt is made to interpret these structural data in terms of the structural evolution of the antibody combining site and the structural basis for the specificity of the binding of antibody to antigen. First, the pertinent crystallographic and sequence data will be presented. Next, the structural studies which reveal the complementarity between antibody and ligand will be described. Then, structural mechanisms by which different combining site structures could be generated will be reviewed.