During the last 15 years, scientists have developed methods that permit the direct mechanical manipulation of individual molecules. Using this approach, they have begun to investigate the effect of force and torque in chemical and biochemical reactions. These studies span from the study of the mechanical properties of macromolecules, to the characterization of molecular motors, to the mechanical unfolding of individual proteins and RNA. Here I present a review of some of our most recent results using mechanical force to unfold individual molecules of RNA. These studies make it possible to follow in real time the trajectory of each molecule as it unfolds and characterize the various intermediates of the reaction. Moreover, if the process takes place reversibly it is possible to extract both kinetic and thermodynamic information from these experiments at the same time that we characterize the forces that maintain the three-dimensional structure of the molecule in solution. These studies bring us closer to the biological unfolding processes in the cell as they simulate in vitro, the mechanical unfolding of RNAs carried out in the cell by helicases. If the unfolding process occurs irreversibly, I show here that single-molecule experiments can still provide equilibrium, thermodynamic information from non-equilibrium data by using recently discovered fluctuation theorems. Such theorems represent a bridge between equilibrium and non-equilibrium statistical mechanics. In fact, first derived in 1997, the first experimental demonstration of the validity of fluctuation theorems was obtained by unfolding mechanically a single molecule of RNA. It is perhaps a sign of the times that important physical results are these days used to extract information about biological systems and that biological systems are being used to test and confirm fundamental new laws in physics.

In recent years it has become clear that different pathways are involved in the process of removing lesions from DNA. In spite of a continuous surveillance of the genetic integrity by repair enzymes, quite often lesions are not eliminated before the portion of the genome where they have been inserted is used for DNA replication or transcription. Actually, the number of unexcised lesions a cell can tolerate without significantly losing its capacity to reproduce is surprising. As an example, human fibroblasts from certain patients with the genetic disease xeroderma pigmentosum (XP)† are virtually unable to excise pyrimidine dimers, the major DNA lesion produced by short-wavelength UV light.

ABC transporters comprise a large, diverse, and ubiquitous superfamily of membrane active transporters. Their core architecture is a dimer of dimers, comprising two transmembrane (TM) domains that bind substrate, and two ATP-binding cassettes, which use the cell's energy currency to couple substrate translocation to ATP hydrolysis. Despite the availability of over a dozen resolved structures and a wealth of biochemical and biophysical data, this field is bedeviled by controversy and long-standing mechanistic questions remain unresolved. The prevailing paradigm for the ABC transport mechanism is the Switch Model, in which the ATP-binding cassettes dimerize upon binding two ATP molecules, and thence dissociate upon sequential ATP hydrolysis. This cycle of nucleotide-binding domain (NBD) dimerization and dissociation is coupled to a switch between inward- or outward facing conformations of a single TM channel; this alternating access enables substrate binding on one face of the membrane and its release at the other. Notwithstanding widespread acceptance of the Switch Model, there is substantial evidence that the NBDs do not separate very much, if at all, and thus physical separation of the ATP cassettes observed in crystallographic structures may be an artefact. An alternative Constant Contact Model has been proposed, in which ATP hydrolysis occurs alternately at the two ATP-binding sites, with one of the sites remaining closed and containing occluded nucleotide at all times. In this model, the cassettes remain in contact and the active sites swing open in an alternately seesawing motion. Whilst the concept of NBD association/dissociation in the Switch Model is naturally compatible with a single alternating-access channel, the asymmetric functioning proposed by the Constant Contact model suggests an alternating or reciprocating function in the TMDs. Here, a new model for the function of ABC transporters is proposed in which the sequence of ATP binding, hydrolysis, and product release in each active site is directly coupled to the analogous sequence of substrate binding, translocation and release in one of two functionally separate substrate translocation pathways. Each translocation pathway functions 180° out of phase. A wide and diverse selection of data for both ABC importers and exporters is examined, and the ability of the Switch and Reciprocating Models to explain the data is compared and contrasted. This analysis shows that not only can the Reciprocating Model readily explain the data; it also suggests straightforward explanations for the function of a number of atypical ABC transporters. This study represents the most coherent and complete attempt at an all-encompassing scheme to explain how these important proteins work, one that is consistent with sound biochemical and biophysical evidence.

Alpha-synuclein (aS) and its aggregation properties are central in the development and spread of Parkinson's disease. Point mutations and multiplications of the SNCA gene encoding aS cause autosomal dominant forms of the disorder. Moreover, protein inclusions found in the surviving neurons of parkinsonian brains consist mainly of a fibrillar form of aS. Aggregates of aS, which form a transient, complex and heterogeneous ensemble, participate in a wide variety of toxic mechanisms that may be amplified by aS spreading among neighbouring neurons. Recently, significant effort has been directed into the study of the aS aggregation process and the impact of aS aggregates on neuron survival. In this review, we present state-of-the-art biophysical studies on the aS aggregation process in vitro and in cellular models. We comprehensively review the new insights generated by the recent biophysical investigations, which could provide a solid basis from which to design future biomedical studies. The diverse cellular models of aS toxicity and their potential use in the biophysical investigation are also discussed.

RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutations in vivo.

1. Introduction 363

2. Motif and sequence design 364

3. Structural and topological constructions 366

4. Nanomechanical devices 367

5. Conclusions, applications and challenges 370

6. Acknowledgments 371

7. References 371

Structural DNA nanotechnology consists of constructing objects, lattices and devices from branched DNA molecules. Branched DNA molecules open the way for the construction of a variety of N-connected motifs. These motifs can be joined by cohesive interactions to produce larger constructs in a bottom-up approach to nanoconstruction. The first objects produced by this approach were stick polyhedra and topological targets, such as knots and Borromean rings. These were followed by periodic arrays with programmable patterns. It is possible to exploit DNA structural transitions and sequence-specific binding to produce a variety of DNA nanomechanical devices, which include a bipedal walker and a machine that emulates the translational capabilities of the ribosome. Much of the promise of this methodology involves the use of DNA to scaffold other materials, such as biological macromolecules, nanoelectronic components, and polymers. These systems are designed to lead to improvements in crystallography, computation and the production of diverse and exotic materials.

Synchrotron radiation is intense electromagnetic radiation with a continuous spectral distribution emitted by high-energy electron (or positron) synchrotrons or storage rings in the visible, vacuum ultraviolet and X-ray regions. For a long time only an unwanted but inevitable by-product of ring accelerators in elementary particle research, synchrotron radiation is now becoming a most widely used tool in atomic, molecular and solid-state spectroscopy, surface physics, structural research on solids, soft X-ray microscopy, lithography. It is not yet clear how large the field of applications really is nor which line of research will prove to be the most fruitful one in the long run.

Ion pumps create ion gradients across cell membranes while consuming light energy or chemical energy. The ion gradients are used by the corresponding cell types for passive-ion transport via ion channels or carriers or for accumulation of nutrients like sugar or amino acids via cotransport systems or antiporters.

A survey of magnetic resonance imaging techniques is presented. Emphasis is put on the basic types of measurement procedures rather than, discussing all variants proposed so far. The general experimental procedure consists of two phases, the preconditioning period and the image formation period. While the preconditioning period determines the image contrast, the image formation process is responsible for image resolution. Means to improve sensitivity and to minimize measurement time are discussed.

About 11 years ago Ford & Benedek (1964) and Cummins, Knable & Yeh (1964) developed some fascinating new light-scattering techniques for the study of macromolecular motions in solution. Enthusiastic researchers have over the years endowed these new methods with a variety of colourful names, including optical mixing or light-beating spectroscopy, heterodyne or homodyne beating, intensity fluctuation spectroscopy, Rayleigh spectroscopy, and quasielastic or dynamic light scattering.

The nucleosome serves as a general gene repressor by the occlusion of regulatory and promoter DNA sequences. Repression is relieved by the SWI/SNF-RSC family of chromatin-remodeling complexes. Research reviewed here has revealed the essential features of the remodeling process.

Photosynthesis in green plants or algae may be represented by an overall equation:

The energy necessary to promote this overall reaction is derived from light through absorption by pigment molecules — chiefly the chlorophylls.

Photosynthesis occurs in chloroplasts - subcellular organelles in which all the chlorophyll pigments are located. The chloroplasts comprise membranous, structures, and can be classified into two types. To the first type belong chloroplasts with appressed stacks of lamellar membranes, termed grana. These chloroplasts occur in mesophyll cells (C3 plants). The second type of chloroplasts are those with lamellar membranes that do not form the grana structures; they occur in bundle sheath cells of maize and other monocotyledons (C4plants, Hatch & Slack, 1970). In algae a greater diversity of structure occurs (Kirk & Tilney-Basset, 1978). Fluorescence microscopy indicates that chlorophyll molecules are localized mainly in the grana membrane regions of mesophyll-type chloroplasts and uniformly throughout the bundle sheath cells (Spencer & Wildman, 1962). Mesophyll chloroplasts are flattened saucer-shaped organelles (20 or more in each cell) of between 5000 and 10000 nm in diameter, and of thickness 1000–2000 nm, whilst the individual grana are each of the order of 300–500 nm in diameter. The available evidence suggests that individual lamellar membranes are arranged to form vesicles, or sacks where the internal space is completely delimited from the external space. These individual closed membrane structures were termed thylakoids (Menke, 1962).

Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins, especially those accomplished with the proteins in phospholipid bilayer environments where they function.

Information on the swimming motion of microscopic calls and the factors which affect it is important to a wide range of disciplines. The functions of the motile apparatus of simpler organisms such as bacteria or algae can provide useful clues on the operation of more complex contractile systems such as muscles. Often celluar motility is a response to conditions external to the cell (e.g. chemotaxis) and, hence, can lead to increased understanding of the cell's sensory capability. On the medical front there is a physical similarity between the flagellar beat of the motile spermatozoa and the ciliary activity of epithelial lining of respiratory and reproductive tracts. Impairment of the motile apparatus can lead to sterility and to a variety of pathological conditions. In animal husbandry, in the artificial insemination industry and in sperm banking estimates of the extent of cellular motility and the fraction of cells which are motile are key quantities which can determmine the fertilization capability of a semen sample.

In principle, there exist two ways to contribute to structure determination of macromolecules by X-ray diffraction: (a) by analysing diffraction data obtained from the crystalline state, and (b) by interpretation of X-ray small-angle scattering from particles in solution.

The brilliant achievements of X-ray crystal-structure analysis of macromolecules, initiated by the works of Perutz on heamoglobin and Kendrew on myoglobin, are well known and it is evident that its detailed elution of secondary, tertiary and quaternary structure cannot be matched by any other means. However, a number of necessary prerequisites for a successful application, as, for example, the availability of well-defined crystals and heavy atom labelled derivatives thereof to surmount the problem of phase determination are not always given.

Altered chromatin structures and dynamics are responsible for a range of human malignancies, among which the status of histone lysine methylation remains of paramount importance. Histone lysine methylation is maintained by the relative activities of sequence-specific methyltransferase (KMT) writers and demethylase (KDM) erasers, with aberrant enzymatic activities or expression profiles closely correlated with multiple human diseases. Hence, targeting these epigenetic enzymes should provide a promising avenue for pharmacological intervention of aberrantly marked sites within the epigenome. Here we present an up-to-date critical evaluation on the development and optimization of potent small molecule inhibitors targeted to histone KMTs and KDMs, with the emphasis on contributions of structural biology to development of epigenetic drugs for therapeutic intervention. We anticipate that ongoing advances in the development of epigenetic inhibitors should lead to novel drugs that site-specifically target KMTs and KDMs, key enzymes responsible for maintenance of the lysine methylation landscape in the epigenome.

Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long–short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction. Just as important, the technique has not failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.

Microtubule proteins have recently been prepared and characterized from a number of diverse sources. Renaud, Rowe & Gibbons (1966, 1968) have obtained a protein from acetone powders of Tetrahymena pyriformis cilia and from isolated outer fibre doublets of this same species. The protein has an actin-like amino acid composition, a minimum subunit weight of 55000, and 7·5 free sulphydryl groups per mole of monomer. Stephens (1968a) has fractionated sea-urchin flagella, following the procedure of Gibbons (1965), to obtain the isolated outer fibres and has found the protein to have properties virtually identical to those of its ciliary counterpart. Both the flagellar and the ciliary outer-fibre proteins contain 1 mole of bound guanine nucleotide per mole of protein subunit (Stephens, Renaud & Gibbons, 1967).