The brain is one of the most highly organized structures in the known universe. It is a biological computer which has evolved over a billion years to program, monitor and control all bodily functions. It is also the organ of knowing, feeling and thinking. To understand how the brain works is perhaps the most difficult of all scientific problems. A scientific theory of the brain would provide a comprehensive understanding of a substantial body of facts on the basis of a few fundamental principles. In this sense, there is no theory of the brain.

Resonance Raman (RR) spectroscopy provides detailed information on the vibrational and electronic properties of biochemical and biological chromophores. The analysis of RR spectra, using for example model compounds or a group frequency approach, enables us to form an accurate structural picture of the chromophore in its natural biological site. Moreover, the insight gained into the electronic states of a biological chromophore can be crucial to our understanding of its function. Thus the RR technique represents a powerful means of eliciting precise structural and electronic data from a coloured species and of focusing upon key aspects of its function. It has even been possible to obtain RR spectra from some natural chromophores in vivo, giving spectra detailed and informative enough to please a spectroscopist from a system complex enough to satisfy a biologist.

About 3 billion years ago an enzyme emerged which would dramatically change the chemical composition of our planet and set in motion an unprecedented explosion in biological activity. This enzyme used solar energy to power the thermodynamically and chemically demanding reaction of water splitting. In so doing it provided biology with an unlimited supply of reducing equivalents needed to convert carbon dioxide into the organic molecules of life while at the same time produced oxygen to transform our planetary atmosphere from an anaerobic to an aerobic state. The enzyme which facilitates this reaction and therefore underpins virtually all life on our planet is known as Photosystem II (PSII). It is a pigment-binding, multisubunit protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Today we have detailed understanding of the structure and functioning of this key and unique enzyme. The journey to this level of knowledge can be traced back to the discovery of oxygen itself in the 18th-century. Since then there has been a sequence of mile stone discoveries which makes a fascinating story, stretching over 200 years. But it is the last few years that have provided the level of detail necessary to reveal the chemistry of water oxidation and O–O bond formation. In particular, the crystal structure of the isolated PSII enzyme has been reported with ever increasing improvement in resolution. Thus the organisational and structural details of its many subunits and cofactors are now well understood. The water splitting site was revealed as a cluster of four Mn ions and a Ca ion surrounded by amino-acid side chains, of which seven provide direct ligands to the metals. The metal cluster is organised as a cubane structure composed of three Mn ions and a Ca2+ linked by oxo-bonds with the fourth Mn ion attached to the cubane. This structure has now been synthesised in a non-protein environment suggesting that it is a totally inorganic precursor for the evolution of the photosynthetic oxygen-evolving complex. In summary, the overall structure of the catalytic site has given a framework on which to build a mechanistic scheme for photosynthetic dioxygen generation and at the same time provide a blue-print and incentive to develop catalysts for artificial photo-electrochemical systems to split water and generate renewable solar fuels.

1. The ribosome 204

2. Peptide bond formation is catalyzed by RNA 205

3. Characteristics of the uncatalyzed reaction 207

4. Potential catalytic strategies of the ribosome 207

5. Experimental systems 208

6. Substrate binding in the PT center 210

7. Induced fit in the active site 211

8. pH dependence of peptide bond formation 212

9. Reaction with full-length aa-tRNA 214

10. Role of active-site residues 215

11. pH-dependent structural changes of the active site 216

12. Entropic catalysis 217

13. Role of 2′-OH of A76 in P-site tRNA 218

14. Catalysis by proton shuttling 219

15. Plasticity of the active site 220

16. Conclusions 221

17. Acknowledgments 222

18. References 222

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site – the peptidyl transferase center – is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 107-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.

Commercially available electron microscopes routinely provide resolution of some 2–4 Å, as determined on the spacing of crystalline lattices of certain stable, small-molecular substances. On biological material either macromolecules or macromolecular assemblies— ‘biologically significant’ details below some 20 Å have hitherto not been observed.we consider as ‘biologically significant’ those structural details observed or contained in electronmicrographs which are consistent with, or confirmed by, other data obtained from biochemical or functional experiments or by other physical methods (optical, magnetic, electric).

Proteins exposed to ionizing radiation suffer both reversible and irreversible effects. Reversible effects are defined as those which disappear in a short period of time after the removal of the radiation field and without further treatment of the sample. Irreversible effects are those which cause a permanent alteration in the structure of a protein.

The relationship between functional properties of haem proteins, particularly ligand binding and Bohr effect, and associated variations of the tertiary and quaternary structures is one of the main objectives of haem protein research. In this context one aims to get detailed knowledge of the coupling mechanisms which are involved in the transduction of structural changes from the protein to the functional haem group along distinct pathways.

The emergence of functional cooperation between the three main classes of biomolecules – nucleic acids, peptides and lipids – defines life at the molecular level. However, how such mutually interdependent molecular systems emerged from prebiotic chemistry remains a mystery. A key hypothesis, formulated by Crick, Orgel and Woese over 40 year ago, posits that early life must have been simpler. Specifically, it proposed that an early primordial biology lacked proteins and DNA but instead relied on RNA as the key biopolymer responsible not just for genetic information storage and propagation, but also for catalysis, i.e. metabolism. Indeed, there is compelling evidence for such an ‘RNA world’, notably in the structure of the ribosome as a likely molecular fossil from that time. Nevertheless, one might justifiably ask whether RNA alone would be up to the task. From a purely chemical perspective, RNA is a molecule of rather uniform composition with all four bases comprising organic heterocycles of similar size and comparable polarity and pKa values. Thus, RNA molecules cover a much narrower range of steric, electronic and physicochemical properties than, e.g. the 20 amino acid side-chains of proteins. Herein we will examine the functional potential of RNA (and other nucleic acids) with respect to self-replication, catalysis and assembly into simple protocellular entities.

The kinetics of biochemical and biophysical events determined the course of life processes and attracted considerable interest and research. For example, modeling of biological networks and cellular responses relies on the availability of information on rate coefficients. Atomically detailed simulations hold the promise of supplementing experimental data to obtain a more complete kinetic picture. However, simulations at biological time scales are challenging. Typical computer resources are insufficient to provide the ensemble of trajectories at the correct length that is required for straightforward calculations of time scales. In the last years, new technologies emerged that make atomically detailed simulations of rate coefficients possible. Instead of computing complete trajectories from reactants to products, these approaches launch a large number of short trajectories at different positions. Since the trajectories are short, they are computed trivially in parallel on modern computer architecture. The starting and termination positions of the short trajectories are chosen, following statistical mechanics theory, to enhance efficiency. These trajectories are analyzed. The analysis produces accurate estimates of time scales as long as hours. The theory of Milestoning that exploits the use of short trajectories is discussed, and several applications are described.

The dynamics of macromolecules, the subject of this symposium, are most directly studied by simply looking through a microscope and observing the molecular motion. With a microscope, we can resolve the size and shape of large particles, as well as monitor dynamic motion. For smaller particles, particularly single macromolecules, we cannot resolve the size or shape; but it is still possible to observe the motion, if we can make the particles appear as bright points of light sprinkled dilutely over a dark background. Siedentopf & Zsigmondy (1903) demonstrated this fact with a device which came to be called the ultramicroscope.

In all life sciences there can be noted a marked trend toward an increased use of physical instrumentation. Calorimeters, which are instruments for measurements of heat quantities or heat effects, offer one approach in this respect.

Several cases of modifications of functional properties of proteins upon specific interaction with antibodies have been reported in the last few years. The purpose of this paper is to discuss evidence that these effects may be due to conformational changes in the protein molecule.

1. INTRODUCTION 68

1.1 Receptor patterns in the plasma membrane 68

1.2 Different types of receptor patterns 71

2. METHODS TO INVESTIGATE NON-RANDOM RECEPTOR CLUSTERING 73

2.1 Fluorescence resonance energy transfer 73

2.2 Flow cytometric energy transfer measurement 78

2.3 Fluorescence anisotropy and energy transfer 79

2.4 Photobleaching energy transfer on single cells 81

2.5 Two-dimensional mapping of receptor superstructures 82

2.6 Detecting single receptor molecules 85

2.7 Chemical identification of receptor clusters 86

2.8 Electron microscopy 87

2.9 Scanning force microscopy 88

3. CONFORMATIONAL STATES OF RECEPTORS 90

3.1 Multi-subunit receptor structures 90

3.2 Physical parameters influencing conformational states 91

3.3 Chemical interactions and receptor conformations 92

4. ON THE ORIGIN OF NATURALLY OCCURRING RECEPTOR CLUSTERS 93

4.1 Synthesis of receptors and their localization in the plasma membrane 93

4.2 Lipid domain structure of the plasma membrane 94

4.3 The validity of the SingerNicolson model 94

5. CONCLUSIONS 96

6. ACKNOWLEDGEMENTS 96

7. REFERENCES 97

DNA dynamics can only be understood by taking into account its complex mechanical behavior at different length scales. At the micrometer level, the mechanical properties of single DNA molecules have been well-characterized by polymer models and are commonly quantified by a persistence length of 50 nm (~150 bp). However, at the base pair level (~3.4 Å), the dynamics of DNA involves complex molecular mechanisms that are still being deciphered. Here, we review recent single-molecule experiments and molecular dynamics simulations that are providing novel insights into DNA mechanics from such a molecular perspective. We first discuss recent findings on sequence-dependent DNA mechanical properties, including sequences that resist mechanical stress and sequences that can accommodate strong deformations. We then comment on the intricate effects of cytosine methylation and DNA mismatches on DNA mechanics. Finally, we review recently reported differences in the mechanical properties of DNA and double-stranded RNA, the other double-helical carrier of genetic information. A thorough examination of the recent single-molecule literature permits establishing a set of general ‘rules’ that reasonably explain the mechanics of nucleic acids at the base pair level. These simple rules offer an improved description of certain biological systems and might serve as valuable guidelines for future design of DNA and RNA nanostructures.

We review the concept of superresolution optical fluctuation imaging (SOFI), discuss its attributes and trade-offs (in comparison with other superresolution methods), and present superresolved images taken on samples stained with quantum dots, organic dyes, and plasmonic metal nanoparticles. We also discuss the prospects of SOFI for live cell superresolution imaging and for imaging with other (non-fluorescent) contrasts.

Increasing evidence suggests that most proteins occur and function in complexes rather than as isolated entities when embedded in cellular membranes. Nuclear magnetic resonance (NMR) provides increasing possibilities to study structure, dynamics and assembly of such systems. In our review, we discuss recent methodological progress to study membrane–protein complexes (MPCs) by NMR, starting with expression, isotope-labeling and reconstitution protocols. We review approaches to deal with spectral complexity and limited spectral spectroscopic sensitivity that are usually encountered in NMR-based studies of MPCs. We highlight NMR applications in various classes of MPCs, including G-protein-coupled receptors, ion channels and retinal proteins and extend our discussion to protein–protein complexes that span entire cellular compartments or orchestrate processes such as protein transport across or within membranes. These examples demonstrate the growing potential of NMR-based studies of MPCs to provide critical insight into the energetics of protein–ligand and protein–protein interactions that underlie essential biological functions in cellular membranes.

In many important types of observations in biological research, the information provided by the specimens is in the form of photons-quanta of visible light, u.v., or X-rays. The process of observation becomes one of recording this information in useful form, with as high an efficiency as possible. The problem becomes particularly important when for some reason or other the total number or rate of quanta provided by the specimen is small. Examples of such limitations are included in the following: (i) Processes permitting only low-intensity illumination in order not to interfere with the biological processes under observation. (ii) Processes changing very rapidly and requiring rapid sequence recording. (iii) Processes providing only a limited number of photons per event, such as bioluminescence. (iv) Processes in which radioactive tracers are utilized, and observation of radioactivity is desirable at low specific activity or within short time intervals. (v) X-ray diffraction processes where the specimen is weakly diffracting or where the X-ray intensity must be kept low in order not to damage the specimen. (vi) Processes involving the observation of fluorescence, where the intensity is low because of limitations on the amount of tagging material.

When Hodgkin & Huxley (1952) first embarked on the analysis of their voltageclamp data on the ionic currents in the squid giant axon, they hoped to be able to deduce a mechanism from it, but it soon became clear that the electrical data would by themselves yield only very general information about the class of system likely to be involved.

1. How do ribozymes work? 241

2. The hammerhead RNA as a prototype ribozyme 242

2.1 RNA enzymes 242

2.2 Satellite self-cleaving RNAs 242

2.3 Hammerhead RNAs and hammerhead ribozymes 244

3. The chemical mechanism of hammerhead RNA self-cleavage 246

3.1 Phosphodiester isomerization via an SN2(P) reaction 247

3.2 The canonical role of divalent metal ions in the hammerhead ribozyme reaction 251

3.3 The hammerhead ribozyme does not actually require metal ions for catalysis 254

3.4 Hammerhead RNA enzyme kinetics 257

4. Sequence requirements for hammerhead RNA self-cleavage 260

4.1 The conserved core, mutagenesis and functional group modifications 260

4.2 Ground-state vs. transition-state effects 261

5. The three-dimensional structure of the hammerhead ribozyme 262

5.1 Enzyme–inhibitor complexes 262

5.2 Enzyme–substrate complex in the initial state 264

5.3 Hammerhead ribozyme self-cleavage in the crystal 264

5.4 The requirement for a conformational change 265

5.5 Capture of conformational intermediates using crystallographic freeze-trapping 266

5.6 The structure of a hammerhead ribozyme ‘early’ conformational intermediate 267

5.7 The structure of a hammerhead ribozyme ‘later’ conformational intermediate 268

5.8 Is the conformational change pH dependent? 269

5.9 Isolating the structure of the cleavage product 271

5.10 Evidence for and against additional large-scale conformation changes 274

5.11 NMR spectroscopic studies of the hammerhead ribozyme 278

6. Concluding remarks 280

7. Acknowledgements 281

8. References 281

1. How do ribozymes work? 241

The discovery that RNA can be an enzyme (Guerrier-Takada et al. 1983; Zaug & Cech, 1986) has created the fundamental question of how RNA enzymes work. Before this discovery, it was generally assumed that proteins were the only biopolymers that had sufficient complexity and chemical heterogeneity to catalyze biochemical reactions. Clearly, RNA can adopt sufficiently complex tertiary structures that make catalysis possible. How does the three- dimensional structure of an RNA endow it with catalytic activity? What structural and functional principles are unique to RNA enzymes (or ribozymes), and what principles are so fundamental that they are shared with protein enzymes?

An intact living muscle has such a regular structure that it diffracts light or X-rays, thereby providing patterns that contain uniquely valuable information. Interpretation of these patterns is not straightforward, but is helped by light microscopy and electron microscopy, which can often provide similar though less reliable information. At all levels of complexity, from that of the fibrils to that of the molecules, structure in a muscle is orderly. No other natural cell assembly is so suited to study by the diffraction method, and the results obtained in recent years are an outstanding example of how this method can elucidate a biological problem. In contrast to protein crystallography, where the system studied is artificial, muscle can be examined in its natural state, during normal activity. The levels of structure explored as yet in muscle are above that of the atoms in the molecules. Such structure is more commonly investigated by electron microscopy, and the application of the diffraction method to living muscle has provided a valuable check on the preparative artifacts that worry the microscopist. The great complexity of a muscle, as compared with a protein crystal, and the fact that the system is only semi-crystalline, giving a much less detailed diffraction pattern, make the problems of interpretation especially difficult. But a great deal of useful information is available about other properties of muscle and its constituents, and the flourishing state of muscle biology at present is a major factor contributing to the successful application of the diffraction method.