One feature characterizing the transition from prokaryote to eukaryote is the ‘sudden’ appearance of centrioles and their highly structured products, the typical eukaryotic flagella and cilia. These mechanochemical systems appear as fully developed machines, containing some 200 diffierent proteins (Luck et al. 1978) arranged in a remarkably complex organization which has undergone little modification since the advent of the first eukaryotic cells. It is now well established (see, for example, Satir, 1974) that ciliary and flagellar motility is based on a sliding filament mechanism that superficially resembles the far more extensively studied sliding filament system of striated skeletal muscle.The flagellar system, however, appears to be much more complex than the muscle system, because it does not ‘merely’ shorten and generate force, but develops propagating waves and exerts its effects via hydrodynamic interactions with a viscous medium.

Bacterial membranes represent an attractive target for the design of new antibiotics to combat widespread bacterial resistance to traditional inhibitor-based antibiotics. Understanding how antimicrobial peptides (AMPs) and other membrane-active agents attack membranes could facilitate the design of new, effective antimicrobials. AMPs, which are small, gene-encoded host defense proteins, offer a promising basis for the study of membrane-active antimicrobial agents. These peptides are cationic and amphipathic, spontaneously binding to bacterial membranes and inducing transmembrane permeability to small molecules. Yet there are often confusions surrounding the details of the molecular mechanisms of AMPs. Following the doctrine of structure–function relationship, AMPs are often viewed as the molecular scaffolding of pores in membranes. Instead we believe that the full mechanism of AMPs is understandable if we consider the interactions of AMPs with the whole membrane domain, where interactions induce structural transformations of the entire membrane, rather than forming localized molecular structures. We believe that it is necessary to consider the entire soft matter peptide-membrane system as it evolves through several distinct states. Accordingly, we have developed experimental techniques to investigate the state and structure of the membrane as a function of the bound peptide to lipid ratio, exactly as AMPs in solution progressively bind to the membrane and induce structural changes to the entire system. The results from these studies suggest that global interactions of AMPs with the membrane domain are of fundamental importance to understanding the antimicrobial mechanisms of AMPs.

1. Introduction 1

1.1 The ‘band gap’ in silicon 2

2. Principles of CCD detector operation 3

2.1 Direct detection 3

2.2 Electron energy conversion into light 4

2.3 Optical coupling: lens or fibre optics? 6

2.4 Readout speed and comparison with film 8

3. Practical considerations for electron microscopic applications 9

3.1 Sources of noise 9

3.1.1 Dark current noise 9

3.1.2 Readout noise 9

3.1.3 Spurious events due to X-rays or cosmic rays 10

3.2 Efficiency of detection 11

3.3 Spatial resolution and modulation transfer function 12

3.4 Interface to electron microscope 14

3.5 Electron diffraction applications 15

4. Prospects for high-resolution imaging with CCD detectors 18

5. Alternative technologies for electronic detection 23

5.1 Image plates 23

5.2 Hybrid pixel detectors 24

6. References 26

During the past decade charge-coupled device (CCD) detectors have increasingly become the preferred choice of medium for recording data in the electron microscope. The CCD detector itself can be likened to a new type of television camera with superior properties, which makes it an ideal detector for recording very low exposure images. The success of CCD detectors for electron microscopy, however, also relies on a number of other factors, which include its fast response, low noise electronics, the ease of interfacing them to the electron microscope, and the improvements in computing that have made possible the storage and processing of large images.

CCD detectors have already begun to be routinely used in a number of important biological applications such as tomography of cellular organelles (reviewed by Baumeister, 1999), where the resolution requirements are relatively modest. However, in most high- resolution microscopic applications, especially where the goal of the microscopy is to obtain structural information at near-atomic resolution, photographic film has continued to remain the medium of choice. With the increasing interest and demand for high-throughput structure determination of important macromolecular assemblies, it is clearly important to have tools for electronic data collection that bypass the slow and tedious process of processing images recorded on photographic film.

In this review, we present an analysis of the potential of CCD-based detectors to fully replace photographic film for high-resolution electron crystallographic applications.

The intelligent design of new families of DNA-binding antitumour agents must await an understanding at the molecular level of the structure, dynamics and energetics of drug-DNA interactions on currently available systems. Recent progress in this area has been significant and reflects the interplay between footprinting methods that identify the sequence specificity of drug binding, structural approaches that define conformational features in the crystalline and solution states, hydrogen exchange techniques that monitor transient base pair opening and calorimetric methods that partition the enthalpic and entropic contributions to the binding isotherm.

The concept of processes in a living organism as of a most complex enzyme-catalysed system of coupled chemical conversions following both physical and chemical laws, opens up new possibilities of cognizance and control of vital processes.

Peter Läuger introduced me (P.C.J.) to the field of ion-channel electrostatics while I was a sabbatical visitor at Konstanz in 1978–79. Läuger pointed out that the relative conductance of hydrophobic ions through phosphatidyl choline (PC) and glyceryl monooleate (GMO) membranes differed by a factor of about 100 (Hladky & Haydon, 1973), quite consistent with the difference in the water-membrane potential differences in the two systems (Pickar & Benz, 1978). However, cation conductance through gramicidin channels spanning these membranes only differs by a factor of 2–3 (Bamberg et al. 1976). Why? It is the pursuit of an answer to this question which led me into my researches in this field.

Photoreceptor cells are special neurons which convert light to an electrical signal. The absorption of a photon by a rhodopsin molecule triggers a sequence of chemical processes leading to a change in the membrane voltage of the photoreceptor. The mechanism of this light-induced voltage change is different in vertebrates and invertebrates. Light causes a hyperpolarization in vertebrates, but a depolarization in invertebrate photoreceptors.

Only two spectroscopic methods are capable of detecting individual atoms in macromolecular systems, X-ray diffraction in the crystalline state and nuclear magnetic resonance (NMR) in the liquid state. For an enzyme-substrate complex, X-ray diffraction can yield information on the geometric structure at the active site and nuclear magnetic resonance absorption can, in principle, yield information on the electronic structure at the active site and on the conformation of enzyme-substrate complexes. Both types of information are needed for unraveling the mechanism of enzyme catalysis on the molecular level. The exciting successes of X-ray diffraction in delineating active sites are already established; NMR, a comparative latecomer among spectroscopic techniques is just beginning to demonstrate its potentialities. McDonald & Phillips have summarized in their excellent review (1969) the work on direct observation of hydrogen atoms (protons) by NMR spectroscopy which has advanced our knowledge of protein structure. The extensive studies of Jardetzky and his co-workers on the NMR of ribonuclease and its inhibitor complexes has culminated in a suggested mechanism of catalytic action (Roberts et al. 1969).

1. Introduction 345

2. Peptide nucleic acid (PNA) 346

3. ‘Cell penetrating peptides’ (CPPs) 346

4. Endosomal escape 347

5. Cellular delivery of PNA 347

6.In vivobioavailability of PNA 349

7. References 350

Recent results on the cellular delivery of antisense peptide nucleic acids (PNA) via peptide conjugation is briefly discussed, in particular in the context of endosomal entrapment and escape.

The great number of investigations and advanced developments in neurophysiology and psychoacoustics during recent years have extensively increased our knowledge about the frequency analysis of simple sounds in the peripheral auditory system.

New methods have facilitated quantitative measurements of the amplitude of the submicroscopic vibration of a narrow segment of the basilar membrane in anaesthetized animals at physiological sound intensities. The results of these studies have quantitatively confirmed the results of past studies by showing that the basilar membrane has a selectivity with regard to tone frequency. In addition to this, the recent studies have increased our knowledge about the finer details of vibration of the basilar membrane. At the lowest levels used in the recent investigations, i.e. about 70 dB SPL, the selectivity in the 7 kHz region of the basilar membrane was found to be greater than expected on the basis of extrapolation of older data. Moreover, the high frequency slope of the tuning curves of the basilar membrane was found to be particularly steep. The results of these recent studies, furthermore, showed that the basilar membrane vibrates in a non-linear way at intensities within the physiological range. This non-linearity results in a broadening of the selectivity curves of a narrow segment of the basilar membrane when the sound intensity is increased.

Little is known as to how the motion of the basilar membrane is transformed to excitation of the cochlear sensory cells, i.e. the haircells. The excitation may be related to displacement, spatial differentiation or other transformations of the basilar membrane motion. Recording from the interior of mammalian haircells has so far been unsuccessful, and the neural excitatory process within the haircells in the cochlea is as yet practically unknown. Studies of the haircells in the lateral line organ of fish have provided fundamental knowledge about their excitation; since they in many respects resemble those in the mammalian cochlea, the results very probably can be applied to the excitatory process in the mammalian cochlea.

It is well known that excited states may be generated chemically in biological systems as evidencex and by the phenomenon of bioluminescence and it is natural to suspect that they may also be generated and used in dark processes (Szent-Györgyi, 1941; Steele, 1963; Cilento, 1965; White & Wei, 1970; White et al. 1971). Förster (1967) has pointed out that electronic excitation and subsequent transfer processes may occur in biological dark systems despite the fact that the energy available from enzymic processes is too low to excite aromatic amino acids and other biochemical structures. Hastings (1968) suggests that in some organisms light emission is just an alternative to the formation of an active species.

Fluorescence turn-on aptamers, in vitro evolved RNA molecules that bind conditional fluorophores and activate their fluorescence, have emerged as RNA counterparts of the fluorescent proteins. Turn-on aptamers have been selected to bind diverse fluorophores, and they achieve varying degrees of specificity and affinity. These RNA–fluorophore complexes, many of which exceed the brightness of green fluorescent protein and their variants, can be used as tags for visualizing RNA localization and transport in live cells. Structure determination of several fluorescent RNAs revealed that they have diverse, unrelated overall architectures. As most of these RNAs activate the fluorescence of their ligands by restraining their photoexcited states into a planar conformation, their fluorophore binding sites have in common a planar arrangement of several nucleobases, most commonly a G-quartet. Nonetheless, each turn-on aptamer has developed idiosyncratic structural solutions to achieve specificity and efficient fluorescence turn-on. The combined structural diversity of fluorophores and turn-on RNA aptamers has already produced combinations that cover the visual spectrum. Further molecular evolution and structure-guided engineering is likely to produce fluorescent tags custom-tailored to specific applications.

X-ray crystallography enables detailed structural studies of proteins to understand and modulate their function. Conducting crystallographic experiments at cryogenic temperatures has practical benefits but potentially limits the identification of functionally important alternative protein conformations that can be revealed only at room temperature (RT). This review discusses practical aspects of preparing, acquiring, and analyzing X-ray crystallography data at RT to demystify preconceived impracticalities that freeze progress of routine RT data collection at synchrotron sources. Examples are presented as conceptual and experimental templates to enable the design of RT-inspired studies; they illustrate the diversity and utility of gaining novel insights into protein conformational landscapes. An integrative view of protein conformational dynamics enables opportunities to advance basic and biomedical research.

We review recent computational advances in the study of membrane proteins, focusing on those that have at least one transmembrane helix. Transmembrane protein regions are, in many respects, easier to investigate computationally than experimentally, due to the uniformity of their structure and interactions (e.g. consisting predominately of nearly parallel helices packed together) on one hand and presenting the challenges of solubility on the other. We present the progress made on identifying and classifying membrane proteins into families, predicting their structure from amino-acid sequence patterns (using many different methods), and analyzing their interactions and packing. The total result of this work allows us for the first time to begin to think about the membrane protein interactome, the set of all interactions between distinct transmembrane helices in the lipid bilayer.

Whenever fundamental features of living systems and their molecular basis are reviewed, the problem of timing, of time setting or free open-end running times is only marginally on the desk of research agendas, although the finite ageing as one of the features resulting from time markers is known since long. With the discovery of cellular and most important of cellfree oscillatory processes new concepts and experimental techniques were designed to approach these questions more directly leading not only to a better understanding of timing but strongly contributed to concepts for spatial pattern generation. As given in the list of contents major items in the field of intracellular and intercellular periodic reactions are reviewed in Sections 2–7 in terms of specific properties of various systems and in Section 8 in summing important features common to all oscillatory stems in chemistry and biology. Section 9 draws attention to the problem of patterning in the mesoscopic domains of living systems, which is so basic in terms of the volume dimensions specific for the cellular and subcellular reaction compartments in biology. The last chapter sets some marks on urgent problems currently approached by the combined methods of molecular genetics, biochemistry and computer technologies.

1. INTRODUCTION AND HISTORY 122

2. PERIODIC GLYCOLYSIS: TEMPORAL AND SPATIAL OSCILLATIONS 123

2.1 Cell-free glycolysis 123

2.2 Intact cells 128

2.2.1 Yeast 128

2.2.2 Myocytes 130

2.2.3 β-Cells of the islets of pancreas 131

3. CALCIUM OSCILLATIONS 131

3.1 Temporal oscillations 131

3.2 Calcium waves 136

3.3 Physiology 138

4. MICROTUBULE OSCILLATIONS 140

5. THE MITOTIC OSCILLATOR 141

6. DICTYOSTELIUM DISCOIDEUM 144

7. CARDIOMUSCULAR NETWORK 150

8. GENERAL PROPERTIES OF DYNAMIC SPATIAL PATTERNS 152

9. MICROSCOPIC PATTERNS 156

10. PERSPECTIVES 162

11. ACKNOWLEDGEMENTS 163

12. REFERENCES 163

The present review describes the methodology of perturbed angular correlation of gamma rays (PAC) applied to biological problems. A large part of the present review focuses on the application of PAC spectroscopy to the study of coordination geometry for the metal site in zinc enzymes. Applications to conformation and dynamics of biomolecules and the use of the method to identify the intracellular sites of either metals or labelled proteins are also discussed.

The nucleosome serves as a general gene repressor, preventing all initiation of transcription except that which is brought about by specific positive regulatory mechanisms. The positive mechanisms begin with chromatin-remodeling by complexes that slide, disrupt, or otherwise alter the structure and organization of nucleosomes. RSC in yeast and its counterpart PBAF in human cells are the major remodeling complexes for transcription. RSC creates a nucleosome-free region in front of a gene, flanked by strongly positioned +1 and −1 nucleosomes, with the transcription start site typically 10–15 bp inside the border of the +1 nucleosome. RSC also binds stably to nucleosomes harboring regulatory elements and to +1 nucleosomes, perturbing their structures in a manner that partially exposes their DNA sequences. The cryo-electron microscope structure of a RSC–nucleosome complex reveals such a structural perturbation, with the DNA largely unwrapped from the nucleosome and likely interacting with a positively charged surface of RSC. Such unwrapping both exposes the DNA and enables its translocation across the histone octamer of the nucleosome by an ATP-dependent activity of RSC. Genetic studies have revealed additional roles of RSC in DNA repair, chromosome segregation, and other chromosomal DNA transactions. These functions of RSC likely involve the same fundamental activities, DNA unwrapping and DNA translocation.

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105 proteins that is underway, raises expectations for equivalent three dimensional structural databases.

Given the existence of a difference in electrical potential between the interior of a nerve cell and the media surrounding it, where the cytoplasm is some 70 mV negative (Hodgkin, 1958), it must be expected that any positively charged ion to which the cell membrane is permeable is more concentrated in the cell interior. For monovalent cations such as Na and divalent cations such as Ca and Mg this is not the case in the majority of the cells such as the squid giant axon. In other words, nerve cells maintain a lower intracellular concentration of these ions, as compared with their concentration in the extracellular fluid. For Mg, Ca and Na ions, this lower internal concentration must, in the steady state, be effected by some membrane based mechanism which consumes energy.