The scope of this article is to describe a transmission X-ray microscope, possible biological applications of soft X-ray microscopy and preliminary results.

For soft X-ray microscopy of biological specimens the wavelength range of 1–10 nm is best suited. Microscopy in this wavelength range requires intense X-ray sources as well as high-resolution X-ray lenses. Intense X-radiation is provided by the synchrotron radiation of electron and positron storage rings. Suited X-ray lenses are zone plates.

A theoretical treatment of the contrast mechanism and the radiation damage as well as first experiments yield the following results. Firstly, relatively thick (1–10 μm) biological specimens can be investigated. This means that unsectioned dried and even wet cells and cell organelles in a natural state can be examined. Second, it will be possible to resolve cellular aggregates in live cells with a resolution in the region of ≤10nm.

The plant root is a complex system that has evolved under the constraints of a number of functions. It is a pressure-probe that can penetrate the soil; it is a scavenger of nutrients that may be either tightly bound to soil particles or in low concentrations in the soil solution; it is an absorber of water from the soil. The tip of the root contains the region of dividing cells from which the root is formed, and is pushed through the soil by the extension of these cells some 10–20 times as they develop and differentiate.

1. Introduction 255

1.1 General thermodynamics 256

2. Nucleic acid thermodynamics 260

2.1 DNA duplexes 261

2.2 RNA duplexes 263

2.3 Hybrid DNA–RNA duplexes 264

2.4 Hydration 267

2.5 Conformational flexibility 269

2.6 Thermodynamics 272

3. Nucleic acid–ligand interactions 277

3.1 Minor groove binders 278

3.2 DNA intercalators 284

3.3 Triple-helical systems 288

3.3.1 Structures 288

3.3.2 Hydration 291

3.3.3 Thermodynamics 291

4. Conclusions 295

5. Acknowledgements 298

6. References 298

In recent years the availability of large quantities of pure synthetic DNA and RNA has revolutionised the study of nucleic acids, such that it is now possible to study their conformations, dynamics and large-scale properties, and their interactions with small ligands, proteins and other nucleic acids in unprecedented detail. This has led to the (re)discovery of higher order structures such as triple helices and quartets, and also the catalytic activity of RNA contingent on three-dimensional folding, and the extraordinary specificity possible with DNA and RNA aptamers.

Nucleic acids are quite different from proteins, even though they are both linear polymers formed from a small number of monomeric units. The major difference reflects the nature of the linkage between the monomers. The 5′–3′ phosphodiester linkage in nucleic acids carries a permanent negative charge, and affords a relatively large number of degrees of freedom, whereas the essentially rigid planar peptide linkage in proteins is neutral and provides only two degrees of torsional freedom per backbone residue. These two properties conspire to make nucleic acids relatively flexible and less likely to form extensive folded structures. Even when true 3D folded structures are formed from nucleic acids, the topology remains simple, with the anionic phosphates forming the surface of the molecule. Nevertheless, nucleic acids do occur in a variety of structures that includes single strands and high-order duplex, triplex or tetraplex (‘quadruplex’) forms. The principles of biological recognition and the related problem of understanding the forces that stabilise such folded structures are in some respects more straightforward than for proteins, making them attractive model systems for understanding general biophysical problems. This view is aided by the relatively facile chemical synthesis of pure nucleic acids of any desired size and defined sequence, and the ease of incorporation of a wide spectrum of chemically modified bases, sugars and backbone linkers. Such modifications are considerably more difficult to achieve with oligopeptides or proteins.

The RNA degradosome is a massive multi-enzyme assembly that occupies a nexus in RNA metabolism and post-transcriptional control of gene expression in Escherichia coli and many other bacteria. Powering RNA turnover and quality control, the degradosome serves also as a machine for processing structured RNA precursors during their maturation. The capacity to switch between destructive and processing modes involves cooperation between degradosome components and is analogous to the process of RNA surveillance in other domains of life. Recruitment of components and cellular compartmentalisation of the degradosome are mediated through small recognition domains that punctuate a natively unstructured segment within a scaffolding core. Dynamic in conformation, variable in composition and non-essential under certain laboratory conditions, the degradosome has nonetheless been maintained throughout the evolution of many bacterial species, due most likely to its diverse contributions in global cellular regulation. We describe the role of the degradosome and its components in RNA decay pathways in E. coli, and we broadly compare these pathways in other bacteria as well as archaea and eukaryotes. We discuss the modular architecture and molecular evolution of the degradosome, its roles in RNA degradation, processing and quality control surveillance, and how its activity is regulated by non-coding RNA. Parallels are drawn with analogous machinery in organisms from all life domains. Finally, we conjecture on roles of the degradosome as a regulatory hub for complex cellular processes.

Serum lipoproteins are the macromolecular lipid–protein complexes, soluble in aqueous environment, which act as transport vehicles for polar and apolar lipids in the bloodstream. The growing multidisciplinary interest in these systems stems on the one hand from the fact that their physiological functions are intimately related to the pathways of lipid metabolism and catabolism, and on the other hand from their macromolecular nature which renders them accessible to a large variety of experimental approaches for the study of the physical and chemical principles of lipid-protein interaction.

Nearly two decades after Westhof and Michel first proposed that RNA tetraloops may interact with distal helices, tetraloop–receptor interactions have been recognized as ubiquitous elements of RNA tertiary structure. The unique architecture of GNRA tetraloops (N=any nucleotide, R=purine) enables interaction with a variety of receptors, e.g., helical minor grooves and asymmetric internal loops. The most common example of the latter is the GAAA tetraloop–11 nt tetraloop receptor motif. Biophysical characterization of this motif provided evidence for the modularity of RNA structure, with applications spanning improved crystallization methods to RNA tectonics. In this review, we identify and compare types of GNRA tetraloop–receptor interactions. Then we explore the abundance of structural, kinetic, and thermodynamic information on the frequently occurring and most widely studied GAAA tetraloop–11 nt receptor motif. Studies of this interaction have revealed powerful paradigms for structural assembly of RNA, as well as providing new insights into the roles of cations, transition states and protein chaperones in RNA folding pathways. However, further research will clearly be necessary to characterize other tetraloop–receptor and long-range tertiary binding interactions in detail – an important milestone in the quantitative prediction of free energy landscapes for RNA folding.

Biological membranes are composed of mainly lipids and proteins. The physical properties of the lipids, forming a bilayer structure, are of crucial importance for the living cell, since the plasma membrane is the guardian barrier towards the environment. Thus, the functioning cell needs a highly stable lipid bilayer, which depends on molecular packing and orientation properties of the various membrane components (Wieslander et al. 1980). The spatial arrangement of the membrane proteins incorporated in the lipid matrix plays an essential role for the different chemical processes occurring at or within the membrane. Information about molecular orientation and mobility is therefore necessary for unravelling the functional mechanisms of a biological membrane.

In recent years, exciting developments in instrument technology and experimental methodology have advanced the field of magic-angle spinning (MAS) nuclear magnetic resonance (NMR) to new heights. Contemporary MAS NMR yields atomic-level insights into structure and dynamics of an astounding range of biological systems, many of which cannot be studied by other methods. With the advent of fast MAS, proton detection, and novel pulse sequences, large supramolecular assemblies, such as cytoskeletal proteins and intact viruses, are now accessible for detailed analysis. In this review, we will discuss the current MAS NMR methodologies that enable characterization of complex biomolecular systems and will present examples of applications to several classes of assemblies comprising bacterial and mammalian cytoskeleton as well as human immunodeficiency virus 1 and bacteriophage viruses. The body of work reviewed herein is representative of the recent advancements in the field, with respect to the complexity of the systems studied, the quality of the data, and the significance to the biology.

Molecular motors, which use energy from ATP hydrolysis to take nanometer-scale steps with run-lengths on the order of micrometers, have important roles in areas such as transport and mitosis in living organisms. New techniques have recently been developed to measure these small movements at the single-molecule level. In particular, fluorescence imaging has contributed to the accurate measurement of this tiny movement. We introduce three single-molecule fluorescence imaging techniques which can find the position of a fluorophore with accuracy in the range of a few nanometers. These techniques are named after Hollywood animation characters: Fluorescence Imaging with One Nanometer Accuracy (FIONA), Single-molecule High-REsolution Colocalization (SHREC), and Defocused Orientation and Position Imaging (DOPI). We explain new understanding of molecular motors obtained from measurements using these techniques.

Cyanobacteria and plants carry out oxygenic photosynthesis. They use water to generate the atmospheric oxygen we breathe and carbon dioxide to produce the biomass serving as food, feed, fibre and fuel. This paper scans the emergence of structural and mechanistic understanding of oxygen evolution over the past 50 years. It reviews speculative concepts and the stepped insight provided by novel experimental and theoretical techniques. Driven by sunlight photosystem II oxidizes the catalyst of water oxidation, a hetero-metallic Mn4CaO5(H2O)4 cluster. Mn3Ca are arranged in cubanoid and one Mn dangles out. By accumulation of four oxidizing equivalents before initiating dioxygen formation it matches the four-electron chemistry from water to dioxygen to the one-electron chemistry of the photo-sensitizer. Potentially harmful intermediates are thereby occluded in space and time. Kinetic signatures of the catalytic cluster and its partners in the photo-reaction centre have been resolved, in the frequency domain ranging from acoustic waves via infra-red to X-ray radiation, and in the time domain from nano- to milli-seconds. X-ray structures to a resolution of 1.9 Å are available. Even time resolved X-ray structures have been obtained by clocking the reaction cycle by flashes of light and diffraction with femtosecond X-ray pulses. The terminal reaction cascade from two molecules of water to dioxygen involves the transfer of four electrons, two protons, one dioxygen and one water. A rigorous mechanistic analysis is challenging because of the kinetic enslaving at millisecond duration of six partial reactions (4e−, 1H+, 1O2). For the time being a peroxide-intermediate in the reaction cascade to dioxygen has been in focus, both experimentally and by quantum chemistry. Homo sapiens has relied on burning the products of oxygenic photosynthesis, recent and fossil. Mankind's total energy consumption amounts to almost one-fourth of the global photosynthetic productivity. If the average power consumption equalled one of those nations with the highest consumption per capita it was four times greater and matched the total productivity. It is obvious that biomass should be harvested for food, feed, fibre and platform chemicals rather than for fuel.

The predominant protein-centric perspective in protein–DNA-binding studies assumes that the protein drives the interaction. Research focuses on protein structural motifs, electrostatic surfaces and contact potentials, while DNA is often ignored as a passive polymer to be manipulated. Recent studies of DNA topology, the supercoiling, knotting, and linking of the helices, have shown that DNA has the capability to be an active participant in its transactions. DNA topology-induced structural and geometric changes can drive, or at least strongly influence, the interactions between protein and DNA. Deformations of the B-form structure arise from both the considerable elastic energy arising from supercoiling and from the electrostatic energy. Here, we discuss how these energies are harnessed for topology-driven, sequence-specific deformations that can allow DNA to direct its own metabolism.

Applications of physical techniques to studies of membrane structure have increased greatly in the past few years and they have begun to provide more precise structural parameters for membranes and also some indication of the physical states of the molecular constituents in the membranes. Direct measurements of membrane features have been made by electron microscopy and by X-ray diffraction methods. Spectroscopic techniques especially infrared absorption, nuclear magnetic resonance absorption and optical rotatory dispersion and circular dichroism measurements have provided additional data relating to the physical states of the molecular components in the membranes.

Piezoelectricity and pyroelectricity in wool and hair were observed by Martin early in 1941. The piezoelectric effect in wood was investigated in detail by Bazhenov (1961). Both converse and direct effect of shear piezoelectricity in wood was demonstrated by Fukada (1955). Bending piezoelectricity in bone was first discovered by Yasuda (1953). Shear and longitudinal piezoelectricity in bone and tendon was quantitatively investigatged by Fukada & Yasuda (1957, 1964).

Desoxyribosenucleic acid, DNA, and cellulose molecules self-assemble in aqueous systems. This aggregation is the basis of the important functions of these biological macromolecules. Both DNA and cellulose have significant polar and nonpolar parts and there is a delicate balance between hydrophilic and hydrophobic interactions. The hydrophilic interactions related to net charges have been thoroughly studied and are well understood. On the other hand, the detailed roles of hydrogen bonding and hydrophobic interactions have remained controversial. It is found that the contributions of hydrophobic interactions in driving important processes, like the double-helix formation of DNA and the aqueous dissolution of cellulose, are dominating whereas the net contribution from hydrogen bonding is small. In reviewing the roles of different interactions for DNA and cellulose it is useful to compare with the self-assembly features of surfactants, the simplest case of amphiphilic molecules. Pertinent information on the amphiphilic character of cellulose and DNA can be obtained from the association with surfactants, as well as on modifying the hydrophobic interactions by additives.

There is an inherent anisotropy in biological and model membrane systems and this anisotropy has a profound influence on the nuclear magnetic resonance spectra of such systems. For nuclei with a quadrupole moment the quadrupole coupling usually dominates the NMR spectrum, while for nuclei with spin quantum number I = ½ dipolar couplings provides the most important effects. The quadrupole coupling only affects isolated spins and usually gives rise to simple spectra. The dipole interactions on the other hand couple several spins. In a lipid bilayer with a multitude of spin-½ nuclei these couplings can give rise to complicated manybody effects. Thus the interpretation of the NMR spectra of membrane systems poses problems but at the same time there is a lot of information to be gained.

Photosynthesis begins with the absorption of light energy and this absorbed energy is transferred to special sites, termed reaction centres. At these sites, the light energy is transformed into chemical products through an oxidation-reduction reaction that generates the primary reactants, an oxidized pigment molecule (P+) and a reduced electron acceptor (A–) (Clayton, 1972). The subsequent reactions of these species in the dark ultimately results in the formation of chemical products required for the fixation of CO2. In this essay we will discuss the nature of the primary reactants generated in the light reactions of chloroplast photosynthesis, stressing recent advances in the identification and characterization of such reactants.

Optimal stereospecific and regiospecific labeling of proteins with stable isotopes enhances the nuclear magnetic resonance (NMR) method for the determination of the three-dimensional protein structures in solution. Stereo-array isotope labeling (SAIL) offers sharpened lines, spectral simplification without loss of information and the ability to rapidly collect and automatically evaluate the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as before. This review gives an overview of stable isotope labeling methods for NMR spectroscopy with proteins and provides an in-depth treatment of the SAIL technology.

Traditional particle-based simulation strategies are impractical for the study of lipid bilayers and biological membranes over the longest length and time scales (microns, seconds and longer) relevant to cellular biology. Continuum-based models developed within the frameworks of elasticity theory, fluid dynamics and statistical mechanics provide a framework for studying membrane biophysics over a range of mesoscopic to macroscopic length and time regimes, but the application of such ideas to simulation studies has occurred only relatively recently. We review some of our efforts in this direction with emphasis on the dynamics in model membrane systems. Several examples are presented that highlight the prominent role of hydrodynamics in membrane dynamics and we argue that careful consideration of fluid dynamics is key to understanding membrane biophysics at the cellular scale.

The flash-cooling of crystals in macromolecular crystallography has become commonplace. The procedure makes it possible to collect data from much smaller specimens than was the case in the past. Also, flash-cooled crystals are much less prone to radiation damage than their room-temperature counterparts, allowing data to be accumulated over extended periods of time. Notwithstanding the attractiveness of the technique, it does have potential disadvantages. First, better methods need to be developed to prevent damage to crystals on freezing. There is also a risk that structures determined at low temperature may suggest conclusions based on aspects of the structure that are not necessarily relevant at room temperature.

The phenomenon of electro-optic orientation was discovered by John Kerr in 1875 and has been used extensively for determining the optical polarizability anisotropy of small molecules and for high-speed transmission of optical signals. Measurements on biopolymers have been made at least since 1950, but only in the last decade have these yielded definitive structural and physical information. In the course of this review, it should become obvious that among the reasons for this late development is the inherent difficulty of analysing optical data that depend simultaneously on intrinsic optical-structural properties of the molecules, and on their degree of orientation under the conditions of the experiment. The problem has been particularly difficult far biopolymers such as the nucleic acids, whose polarization in an electric field is dependent on their special polyelectrolyte properties. These unique electrostatic properties are an important feature in the interpretation of the experimental observations.