It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

Although a unifying characteristic common to all transient receptor potential (TRP) channel functions remains elusive, they could be described as tetramers formed by subunits with six transmembrane domains and containing cation-selective pores, which in several cases show high calcium permeability. TRP channels constitute a large superfamily of ion channels, and can be grouped into seven subfamilies based on their amino acid sequence homology: the canonical or classic TRPs, the vanilloid receptor TRPs, the melastatin or long TRPs, ankyrin (whose only member is the transmembrane protein 1 [TRPA1]), TRPN after the nonmechanoreceptor potential C (nonpC), and the more distant cousins, the polycystins and mucolipins. Because of their role as cellular sensors, polymodal activation and gating properties, many TRP channels are activated by a variety of different stimuli and function as signal integrators. Thus, how TRP channels function and how function relates to given structural determinants contained in the channel-forming protein has attracted the attention of biophysicists as well as molecular and cell biologists. The main purpose of this review is to summarize our present knowledge on the structure of channels of the TRP ion channel family. In the absence of crystal structure information for a complete TRP channel, we will describe important protein domains present in TRP channels, structure–function mutagenesis studies, the few crystal structures available for some TRP channel modules, and the recent determination of some TRP channel structures using electron microscopy.

The application of magnetic resonance techniques to biological systems has permitted a detailed study of the nature of the active sites of many proteins that had not been possible previously. Among these is the whole class of iron—sulphur proteins which have been implicated as electron transport proteins in a variety of fundamental processes: photosynthesis, hydroxylation and nitrogen fixation to name but a few.

The single-iron proteins in this class, the rubredoxins, have been studied extensively by chemical, spectroscopic and X-ray crystallographic techniques (Lovenberg, 1973), and the active site is composed of a single iron atom bound in a distorted tetrahedron of cysteine sulphur ligands. The iron is high-spin ferric in the oxidized state and high-spin ferrous in the reduced state. This structure is shown in Fig. I (α).

Studies of electron-transfer reactions of redox proteins have, in recent years, attracted widespread interest and attention. Progress has been evident from both physical and biological standpoints, with the increasing availability of three-dimensional structural data for many small electron-transfer proteins prompting a variety of systematic investigations (Isied, 1985). Most recently, attention has been directed towards questions concerning the elementary transfer of electrons between spatially remote redox sites, and the nature of protein–protein interactions which, for intermolecular processes, stabilize specific precursor complexes which may be optimally juxtaposed for electron-transfer. These and other issues, including the necessary reversibility of protein interfacial interactions and the dynamic properties of proteins as carriers of electrons in biological electron-transport systems, are now being addressed in the rapidly emerging field of direct (unmediated) protein electrochemistry. It is our intention in this article to discuss developments made in this area and highlight points which we believe to have the most bearing on our current understanding of diffusion-dominated, protein-mediated electron transport at electrode surfaces. First we shall outline some basic considerations which are best considered with reference to homogeneous systems.

1. Chaperonin action – an overview 230

2. Polypeptide binding – an essential action 235

3. Recognition of non-native polypeptide – role of hydrophobicity 236

4. Crystallographic analyses of peptide binding 237

5. Topology and secondary and tertiary structure of bound substrate polypeptide – fluorescence, hydrogen exchange and NMR studies 239

6. Binding by GroEL associated with a putative unfolding action 242

7. A potential action of substrate unfolding driven by ATP/GroES binding 245

8. Folding in theciscavity 247

9. GroEL–GroES-mediated folding of larger substrate proteins by atransmechanism 249

10. Prospects for resolving the conformations and fate of polypeptide in the chaperonin reaction 251

11. References 252

Chaperonins are megadalton ring assemblies that mediate essential ATP-dependent assistance of protein folding to the native state in a variety of cellular compartments, including the mitochondrial matrix, the eukaryotic cytosol, and the bacterial cytoplasm. Structural studies of the bacterial chaperonin, GroEL, both alone and in complex with its co-chaperonin, GroES, have resolved the states of chaperonin that bind and fold non-native polypeptides. Functional studies have resolved the action of ATP binding and hydrolysis in driving the GroEL–GroES machine through its folding-active and binding-active states, respectively. Yet the exact fate of substrate polypeptide during these steps is only poorly understood. For example, while binding involves multivalent interactions between hydrophobic side-chains facing the central cavity of GroEL and exposed hydrophobic surfaces of the non-native protein, the structure of any polypeptide substrate while bound to GroEL remains unknown. It is also unclear whether binding to an open GroEL ring is accompanied by structural changes in the non-native substrate, in particular whether there is an unfolding action. As a polypeptide-bound ring becomes associated with GroES, do the large rigid-body movements of the GroEL apical domains serve as another source of a potential unfolding action? Regarding the encapsulated folding-active state, how does the central cavity itself influence the folding trajectory of a substrate? Finally, how do GroEL and GroES serve, as recently recognized, to assist the folding of substrates too large to be encapsulated inside the machine? Here, such questions are addressed with the findings available to date, and means of further resolving the states of chaperonin-associated polypeptide are discussed.

1. Triple-helical nucleic acids 89

1.1 History 89

1.2 Use of oligomers in triplex formation 90

2. Modes of triplex formation 90

2.1 Intermolecular triplexes 90

2.2 Intramolecular triplexes (H-DNA) 92

2.3 R-DNA (recombination DNA) 92

2.4 PNA (peptide nucleic acids) 93

3. Triplex structural models 93

3.1 YR-Y triplexes 94

3.2 GT-A base triplets 94

3.3 TC-G base triplets 94

3.4 TA-T and C+G-C base triplets 94

3.5 RR-Y triplexes 94

4. Modifications of TFOs 95

4.1 Backbone modification of oligonucleotides 95

4.2 Modification of the ribose in oligonucleotides 96

4.3 Base modification of oligonucleotides 97

5. Gene targeting and modification via triplex technology 98

5.1 Transcription and replication inhibition 99

5.2 TFO-directed mutagenesis 99

5.3 TFO-induced recombination 100

5.4 Future challenges in triplex-directed genome modification 100

6. References 101

The first description of triple-helical nucleic acids was by Felsenfeld and Rich in 1957 (Felsenfeld et al. 1957). While studying the binding characteristics of polyribonucleotides by fiber diffraction studies, they determined that polyuridylic acid [poly(U)] and polyadenylic acid [poly(A)] strands were capable of forming a stable complex of poly(U) and poly(A) in a 2:1 ratio. It was therefore concluded that the nucleic acids must be capable of forming a helical three-stranded structure. The formation of the three-stranded complex was preferred over duplex formation in the presence of divalent cations (e.g. 10 mm MgCl2). The reaction was quite specific, since the (U-A) molecule did not react with polycytidylic acid [(poly(C)], polyadenylic acid or polyinosinic acid [(poly(I)] (Felsenfeld et al. 1957). It was later found that poly(dT-dC) and poly(dG-dA) also have the capacity to form triple-stranded structures (Howard & Miles, 1964; Michelson & Monny, 1967). Other triple helical combinations of polynucleotide strands were identified from X-ray fiber-diffraction studies including, (A)n.2(I)n and (A)n.2(T)n (Arnott & Selsing, 1974). X-ray diffraction patterns of triple-stranded fibers of poly(A).2poly(U) and poly(dA).2poly(dT) showed an A-form conformation of the Watson–Crick strands. The third strand was bound in a parallel orientation to the purine strand by Hoogsteen hydrogen bonds (Hoogsteen, 1959; Arnott & Selsing, 1974). In 1968, the first potential biological role of these structures was identified by Morgan & Wells (1968). Using an in vitro assay, they found that transcription by E. coli RNA polymerase was inhibited by an RNA third strand. Thus, the recent developments identifying the potential of triplex formation for gene regulation and genome modification came more than 20 years after this first study of transcription inhibition by triplex formation.

The electron microscope has contributed deep insights into biological structure since its invention nearly 80 years ago. Advances in instrumentation and methodology in recent decades have now enabled electron tomography to become the highest resolution three-dimensional (3D) imaging technique available for unique objects such as cells. Cells can be imaged either plastic-embedded or frozen-hydrated. Then the series of projection images are aligned and back-projected to generate a 3D reconstruction or ‘tomogram’. Here, we review how electron tomography has begun to reveal the molecular organization of cells and how the existing and upcoming technologies promise even greater insights into structural cell biology.

Molybednum-containing enzymes (Coughlan, 1980; Spiro, 1985) occupy a significant place in the development of the field now termed inorganic biochemistry. The importance of the metal as a biological trace element depends on its involvement in the known, and perhaps other as yet unknown, molybdoenzymes. That it plays a role in biological nitrogen fixation, the process whereby the enzyme nitrogenase in the root nodules of plants converts atmospheric nitrogen into ammonia, was recognized in the 1930s. The metal is also a constituent of a variety of other enzymes, having first been found in a mammalian enzyme, xanthine oxidase, in the 1950s.

We assess the progress in biomolecular modeling and simulation, focusing on structure prediction and dynamics, by presenting the field's history, metrics for its rise in popularity, early expressed expectations, and current significant applications. The increases in computational power combined with improvements in algorithms and force fields have led to considerable success, especially in protein folding, specificity of ligand/biomolecule interactions, and interpretation of complex experimental phenomena (e.g. NMR relaxation, protein-folding kinetics and multiple conformational states) through the generation of structural hypotheses and pathway mechanisms. Although far from a general automated tool, structure prediction is notable for proteins and RNA that preceded the experiment, especially by knowledge-based approaches. Thus, despite early unrealistic expectations and the realization that computer technology alone will not quickly bridge the gap between experimental and theoretical time frames, ongoing improvements to enhance the accuracy and scope of modeling and simulation are propelling the field onto a productive trajectory to become full partner with experiment and a field on its own right.

The majority of studies of the living cell rely on capturing images using fluorescence microscopy. Unfortunately, for centuries, diffraction of light was limiting the spatial resolution in the optical microscope: structural and molecular details much finer than about half the wavelength of visible light (~200 nm) could not be visualized, imposing significant limitations on this otherwise so promising method. The surpassing of this resolution limit in far-field microscopy is currently one of the most momentous developments for studying the living cell, as the move from microscopy to super-resolution microscopy or ‘nanoscopy’ offers opportunities to study problems in biophysical and biomedical research at a new level of detail. This review describes the principles and modalities of present fluorescence nanoscopes, as well as their potential for biophysical and cellular experiments. All the existing nanoscopy variants separate neighboring features by transiently preparing their fluorescent molecules in states of different emission characteristics in order to make the features discernible. Usually these are fluorescent ‘on’ and ‘off’ states causing the adjacent molecules to emit sequentially in time. Each of the variants can in principle reach molecular spatial resolution and has its own advantages and disadvantages. Some require specific transitions and states that can be found only in certain fluorophore subfamilies, such as photoswitchable fluorophores, while other variants can be realized with standard fluorescent labels. Similar to conventional far-field microscopy, nanoscopy can be utilized for dynamical, multi-color and three-dimensional imaging of fixed and live cells, tissues or organisms. Lens-based fluorescence nanoscopy is poised for a high impact on future developments in the life sciences, with the potential to help solve long-standing quests in different areas of scientific research.

Despite continuing advances in the development of macromolecules, including peptides, proteins, and oligonucleotides, for therapeutic purposes, the successful application of these hydrophilic molecules has so far been hampered by their inability to efficiently traverse the cellular plasma membrane. The discovery of a class of peptides (cell-penetrating peptides, CPPs) with the ability to mediate the non-invasive and efficient import of a whole host of cargoes, both in vitro and in vivo, has provided a new means by which the problem associated with cellular delivery can be circumvented. A complete understanding of the translocation mechanism(s) of CPPs has so far proven elusive. Initial studies indicated an ATP-independent, non-endocytotic mechanism, dependent on direct peptide–membrane interactions, making it an enticing challenge from a biophysical point of view. However, recent evidence cast doubt on many of the earlier results, and led to a re-evaluation of the translocation mechanism of CPPs. In this review a brief history of the field will be given, followed by an introduction to some of the better known and more widely used CPPs, including some of their current applications, and finally a discussion of the translocation mechanism(s) and the controversies surrounding it.

The idea that certain drugs and neurotransmitters produce their effects by combining with specific receptors was first clearly expressed by Langley (1905) on the basis of the selective and localized effect of nicotine on striated muscle fibres. In 1914, Langley published a paper in which the antagonism between ‘curari’ and nicotine was analysed and measured as the ratio by which the nicotine concentration had to be increased in order to produce a standard response in the presence of tubocurarine. It was clear that Langley had in mind the idea of competition between nicotine and curare for the receptor sites and it was surprising that he did not formulatethe theory quantitatively, particularly since Hill, working in Langley's laboratory in 1909, published a mathematical analysis of the action of nicotine on frog muscle giving kinetic and equilibrium equations based on the law of mass action, which could easily have been extended to give an account of competitive antagonism. Barger & Dale (1910) did not favour the idea of specific receptors for sympathomimetic amines.

The atomic force microscope (AFM) was invented by Binnig, Quate and Gerberless than 10 years ago (Binnig et al. 1986). In their first prototype, a piece of goldfoil was used as the cantilever, with a crushed diamond tip mounted at the end. On the back of the cantilever, a tunnelling junction was used to monitor the deflection of the cantilever (the gold-foil) when the specimen was scanned with the tip in contact with the surface. Thus, the surface topography of the specimen was obtained with a resolution critically dependent on the sharpness of the tip provided the deformation of the specimen was not serious. Even with such a crude set-up, they managed to obtain a lateral resolution of ˜ 30 Å and a vertical resolution of better than 1 Å on an amorphous A12O3 surface. The operating principle of such an instrument is deceptively simple. However, such an arrangement was inconvenient for routine operations and unsuitable for imaging hydrated specimens, because the tunnelling junction is easily contaminated in air and works poorly in aqueous solutions (Alexander et al. 1989). As a result, the application of this type of AFM to biological samples was rare (Engel, 1991).

The mechanism of energy transduction in biological membranes is a fascinating unsolved problem in biology. It has been recognized for some time that the driving force for a variety of seemingly unrelated phenomena (e.g. secondary active transport, oxidative phosphorylation, rotation of the bacterial flagellar motor) is a bulk-phase, transmembrane electrochemical ion gradient. However, insight into the molecular mechanisms by which free energy stored in such gradients is transduced into work or into chemical energy is just beginning. Nonetheless, gene sequencing and analyses of deduced amino-acid sequences suggest that many biological machines involved in energy transduction, secondary transport proteins in particular (Henderson, 1990; Marger & Saier, 1993), fall into families encompassing proteins from archaea to the mammalian central nervous system, thereby raising the possibility that the members may have common basic structural features and mechanisms. In addition, many of these proteins play important roles in human disease (e.g. diabetes mellitus, glucose/galactose malabsorption, some forms of drug abuse, stroke, antibiotic resistance), as well as the mechanism of action of certain psychotropic drugs.

The focus of this review is on recent observations with a specific secondary transport protein, the lactose permease (lac permease) of Escherichia coli, as a representative of a huge number of proteins that catalyse similar reactions in virtually all biological membranes.

Energy landscape theories have provided a common ground for understanding the protein folding problem, which once seemed to be overwhelmingly complicated. At the same time, the native state was found to be an ensemble of interconverting states with frustration playing a more important role compared to the folding problem. The landscape of the folded protein – the native landscape – is glassier than the folding landscape; hence, a general description analogous to the folding theories is difficult to achieve. On the other hand, the native basin phase volume is much smaller, allowing a protein to fully sample its native energy landscape on the biological timescales. Current computational resources may also be used to perform this sampling for smaller proteins, to build a ‘topographical map’ of the native landscape that can be used for subsequent analysis. Several major approaches to representing this topographical map are highlighted in this review, including the construction of kinetic networks, hierarchical trees and free energy surfaces with subsequent structural and kinetic analyses. In this review, we extensively discuss the important question of choosing proper collective coordinates characterizing functional motions. In many cases, the substates on the native energy landscape, which represent different functional states, can be used to obtain variables that are well suited for building free energy surfaces and analyzing the protein's functional dynamics. Normal mode analysis can provide such variables in cases where functional motions are dictated by the molecule's architecture. Principal component analysis is a more expensive way of inferring the essential variables from the protein's motions, one that requires a long molecular dynamics simulation. Finally, the two popular models for the allosteric switching mechanism, ‘preexisting equilibrium’ and ‘induced fit’, are interpreted within the energy landscape paradigm as extreme points of a continuum of transition mechanisms. Some experimental evidence illustrating each of these two models, as well as intermediate mechanisms, is presented and discussed.

Several reviews have been published recently on the toxicity of heavy metals, but few of these have made any reference to the influence of the nutritional state of animals upon their tolerance of heavy metals. Furthermore, the clinical and metabolic changes occurring as a consequence of increased dietary intake of heavy metals are extremely dependent on factors such as the mineral composition of the diet and nature of the protein source. These aspects will be given particular attention in this review.

It has been known for many years that there are two distinct ways of designing an electron microscope.

Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein–ligand binding, especially the drug recognition of GPCRs.

Nearly 50 years since its potential as a fluorescent base analogue was first recognized, 2-aminopurine (2AP) continues to be the most widely used fluorescent probe of DNA structure and the perturbation of that structure by interaction with enzymes and other molecules. In this review, we begin by considering the origin of the dramatic and intriguing difference in photophysical properties between 2AP and its structural isomer, adenine; although 2AP differs from the natural base only in the position of the exocyclic amine group, its fluorescence intensity is one thousand times greater. We then discuss the mechanism of interbase quenching of 2AP fluorescence in DNA, which is the basis of its use as a conformational probe but remains imperfectly understood. There are hundreds of examples in the literature of the use of changes in the fluorescence intensity of 2AP as the basis of assays of conformational change; however, in this review we will consider in detail only a few intensity-based studies. Our primary aim is to highlight the use of time-resolved fluorescence measurements, and the interpretation of fluorescence decay parameters, to explore the structure and dynamics of DNA. We discuss the salient features of the fluorescence decay of 2AP when incorporated in DNA and review the use of decay measurements in studying duplexes, single strands and other structures. We survey the use of 2AP as a probe of DNA-enzyme interaction and enzyme-induced distortion, focusing particularly on its use to study base flipping and the enhanced mechanistic insights that can be gained by a detailed analysis of the decay parameters, rather than merely monitoring changes in fluorescence intensity. Finally we reflect on the merits and shortcomings of 2AP and the prospects for its wider adoption as a fluorescence-decay-based probe.