Biological electron transfers often occur between metal-containing cofactors that are separated by very large molecular distances. Employing photosensitizer-modified iron and copper proteins, we have shown that single-step electron tunneling can occur on nanosecond to microsecond timescales at distances between 15 and 20 Å. We also have shown that charge transport can occur over even longer distances by hole hopping (multistep tunneling) through intervening tyrosines and tryptophans. In this perspective, we advance the hypothesis that such hole hopping through Tyr/Trp chains could protect oxygenase, dioxygenase, and peroxidase enzymes from oxidative damage. In support of this view, by examining the structures of P450 (CYP102A) and 2OG-Fe (TauD) enzymes, we have identified candidate Tyr/Trp chains that could transfer holes from uncoupled high-potential intermediates to reductants in contact with protein surface sites.

Detailed understanding of the action of biological molecular machines must overcome the challenge of gaining a clear knowledge of the corresponding free-energy landscape. An example for this is the elucidation of the nature of converting chemical energy to torque and work in the rotary molecular motor of F1-ATPase. A major part of the challenge involves understanding the rotary–chemical coupling from a non-phenomenological structure/energy description. Here we focused on using a coarse-grained model of F1-ATPase to generate a structure-based free-energy landscape of the rotary–chemical process of the whole system. In particular, we concentrated on exploring the possible impact of the position of the catalytic dwell on the efficiency and torque generation of the molecular machine. It was found that the experimentally observed torque can be reproduced with landscapes that have different positions for the catalytic dwell on the rotary–chemical surface. Thus, although the catalysis is undeniably required for torque generation, the experimentally observed position of the catalytic dwell at 80° might not have a clear advantage for the force generation by F1-ATPase. This further implies that the rotary–chemical couplings in these biological motors are quite robust and their efficiencies do not depend explicitly on the position of the catalytic dwells. Rather, the specific positioning of the dwells with respect to the rotational angle is a characteristic arising due to the structural construct of the molecular machine and might not bear any clear connection to the thermodynamic efficiency for the system.

To date, most antibodies from combinatorial libraries have been selected purely on the basis of binding. However, new methods now allow selection on the basis of function in animal cells. These selected agonist antibodies have given new insights into the important problem of signal transduction. Remarkably, when some antibodies bind to a given receptor they induce a cell fate that is different than that induced by the natural agonist to the same receptor. The fact that receptors can be functionally pleiotropic may yield new insights into the important problem of signal transduction.

We describe the development of a chemical process based on the CuAAC reaction (click chemistry) to ligate DNA strands and produce an unnatural triazole backbone linkage. The chemical reaction is templated by a complementary DNA splint which accelerates the reaction and provides the required specificity. The resultant 1,4-triazole linkage is read through by DNA and RNA polymerases and is biocompatible in bacterial and human cells. This work has implications for the synthesis of chemically modified genes and other large modified DNA and RNA constructs.

Copper (Cu) is an essential transition metal providing activity to key enzymes in the human body. To regulate the levels and avoid toxicity, cells have developed elaborate systems for loading these enzymes with Cu. Most Cu-dependent enzymes obtain the metal from the membrane-bound Cu pumps ATP7A/B in the Golgi network. ATP7A/B receives Cu from the cytoplasmic Cu chaperone Atox1 that acts as the cytoplasmic shuttle between the cell membrane Cu importer, Ctr1 and ATP7A/B. Biological, genetic and structural efforts have provided a tremendous amount of information for how the proteins in this pathway work. Nonetheless, basic mechanistic-biophysical questions (such as how and where ATP7A/B receives Cu, how ATP7A/B conformational changes and domain–domain interactions facilitate Cu movement through the membrane, and, finally, how target polypeptides are loaded with Cu in the Golgi) remain elusive. In this perspective, unresolved inquiries regarding ATP7A/B mechanism will be highlighted. The answers are important from a fundamental view, since mechanistic aspects may be common to other metal transport systems, and for medical purposes, since many diseases appear related to Cu transport dysregulation.

Nuclear magnetic resonance is the most important form of molecular spectroscopy in chemistry and biochemistry but it is normally blind to chirality. It was predicted in 2004 that precessing nuclear spins in chiral molecules in a liquid in a strong magnetic field induce a rotating electric polarization that is of opposite sign for enantiomers. This polarization arises from the distortion of the electronic structure by the nuclear magnetic moment in the presence of the strong magnetic field and is equivalent to the linear effect of an electric field on the nuclear shielding tensor. The polarization is strongly enhanced in dipolar molecules through the partial orientation of the permanent dipole through the antisymmetric part of the nuclear magnetic shielding tensor. Alternatively, an applied electric field will induce a chirally sensitive magnetization perpendicular to the field and to the nuclear spin. Progress towards the experimental realization of chiral discrimination by NMR is assessed.

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.

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.

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.

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.

This is a tour of a physical chemist through 65 years of protein chemistry from the time when emphasis was placed on the determination of the size and shape of the protein molecule as a colloidal particle, with an early breakthrough by James Sumner, followed by Linus Pauling and Fred Sanger, that a protein was a real molecule, albeit a macromolecule. It deals with the recognition of the nature and importance of hydrogen bonds and hydrophobic interactions in determining the structure, properties, and biological function of proteins until the present acquisition of an understanding of the structure, thermodynamics, and folding pathways from a linear array of amino acids to a biological entity. Along the way, with a combination of experiment and theoretical interpretation, a mechanism was elucidated for the thrombin-induced conversion of fibrinogen to a fibrin blood clot and for the oxidative-folding pathways of ribonuclease A. Before the atomic structure of a protein molecule was determined by x-ray diffraction or nuclear magnetic resonance spectroscopy, experimental studies of the fundamental interactions underlying protein structure led to several distance constraints which motivated the theoretical approach to determine protein structure, and culminated in the Empirical Conformational Energy Program for Peptides (ECEPP), an all-atom force field, with which the structures of fibrous collagen-like proteins and the 46-residue globular staphylococcal protein A were determined. To undertake the study of larger globular proteins, a physics-based coarse-grained UNited-RESidue (UNRES) force field was developed, and applied to the protein-folding problem in terms of structure, thermodynamics, dynamics, and folding pathways. Initially, single-chain and, ultimately, multiple-chain proteins were examined, and the methodology was extended to protein–protein interactions and to nucleic acids and to protein–nucleic acid interactions. The ultimate results led to an understanding of a variety of biological processes underlying natural and disease phenomena.

Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer ‘sandwich’ architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-1-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production.

Myriad biological processes proceed through states that defy characterization by conventional atomic-resolution structural biological methods. The invisibility of these ‘dark’ states can arise from their transient nature, low equilibrium population, large molecular weight, and/or heterogeneity. Although they are invisible, these dark states underlie a range of processes, acting as encounter complexes between proteins and as intermediates in protein folding and aggregation. New methods have made these states accessible to high-resolution analysis by nuclear magnetic resonance (NMR) spectroscopy, as long as the dark state is in dynamic equilibrium with an NMR-visible species. These methods – paramagnetic NMR, relaxation dispersion, saturation transfer, lifetime line broadening, and hydrogen exchange – allow the exploration of otherwise invisible states in exchange with a visible species over a range of timescales, each taking advantage of some unique property of the dark state to amplify its effect on a particular NMR observable. In this review, we introduce these methods and explore two specific techniques – paramagnetic relaxation enhancement and dark state exchange saturation transfer – in greater detail.

Most biological processes require the production and degradation of proteins, a task that weighs heavily on the cell. Mutations that compromise the conformational stability of proteins place both specific and general burdens on cellular protein homeostasis (proteostasis) in ways that contribute to numerous diseases. Efforts to elucidate the chain of molecular events responsible for diseases of protein folding address one of the foremost challenges in biomedical science. However, relatively little is known about the processes by which mutations prompt the misfolding of α-helical membrane proteins, which rely on an intricate network of cellular machinery to acquire and maintain their functional structures within cellular membranes. In this review, we summarize the current understanding of the physical principles that guide membrane protein biogenesis and folding in the context of mammalian cells. Additionally, we explore how pathogenic mutations that influence biogenesis may differ from those that disrupt folding and assembly, as well as how this may relate to disease mechanisms and therapeutic intervention. These perspectives indicate an imperative for the use of information from structural, cellular, and biochemical studies of membrane proteins in the design of novel therapeutics and in personalized medicine.

The voltage sensitive domain (VSD) is a pivotal structure of voltage-gated ion channels (VGICs) and plays an essential role in the generation of electrochemical signals by neurons, striated muscle cells, and endocrine cells. The VSD is not unique to VGICs. Recent studies have shown that a VSD regulates a phosphatase. Similarly, Hv1, a voltage-sensitive protein that lacks an apparent pore domain, is a self-contained voltage sensor that operates as an H+ channel.

VSDs are formed by four transmembrane helices (S1–S4). The S4 helix is positively charged due to the presence of arginine and lysine residues. It is surrounded by two water crevices that extend into the membrane from both the extracellular and intracellular milieus. A hydrophobic septum disrupts communication between these water crevices thus preventing the permeation of ions. The septum is maintained by interactions between the charged residues of the S4 segment and the gating charge transfer center. Mutating the charged residue of the S4 segment allows the water crevices to communicate and generate gating pore or omega pore. Gating pore currents have been reported to underlie several neuronal and striated muscle channelopathies. Depending on which charged residue on the S4 segment is mutated, gating pores are permeant either at depolarized or hyperpolarized voltages. Gating pores are cation selective and seem to converge toward Eisenmann's first or second selectivity sequences. Most gating pores are blocked by guanidine derivatives as well as trivalent and quadrivalent cations. Gating pores can be used to study the movement of the voltage sensor and could serve as targets for novel small therapeutic molecules.

Biomolecules are the prime information processing elements of living matter. Most of these inanimate systems are polymers that compute their own structures and dynamics using as input seemingly random character strings of their sequence, following which they coalesce and perform integrated cellular functions. In large computational systems with finite interaction-codes, the appearance of conflicting goals is inevitable. Simple conflicting forces can lead to quite complex structures and behaviors, leading to the concept of frustration in condensed matter. We present here some basic ideas about frustration in biomolecules and how the frustration concept leads to a better appreciation of many aspects of the architecture of biomolecules, and especially how biomolecular structure connects to function by means of localized frustration. These ideas are simultaneously both seductively simple and perilously subtle to grasp completely. The energy landscape theory of protein folding provides a framework for quantifying frustration in large systems and has been implemented at many levels of description. We first review the notion of frustration from the areas of abstract logic and its uses in simple condensed matter systems. We discuss then how the frustration concept applies specifically to heteropolymers, testing folding landscape theory in computer simulations of protein models and in experimentally accessible systems. Studying the aspects of frustration averaged over many proteins provides ways to infer energy functions useful for reliable structure prediction. We discuss how frustration affects folding mechanisms. We review here how the biological functions of proteins are related to subtle local physical frustration effects and how frustration influences the appearance of metastable states, the nature of binding processes, catalysis and allosteric transitions. In this review, we also emphasize that frustration, far from being always a bad thing, is an essential feature of biomolecules that allows dynamics to be harnessed for function. In this way, we hope to illustrate how Frustration is a fundamental concept in molecular biology.

The past decade has witnessed significant developments in molecular biology techniques, fluorescent labeling, and super-resolution microscopy, and together these advances have vastly increased our quantitative understanding of the cell. This detailed knowledge has concomitantly opened the door for biophysical modeling on a cellular scale. There have been comprehensive models produced describing many processes such as motility, transport, gene regulation, and chemotaxis. However, in this review we focus on a specific set of phenomena, namely cell polarization, F-actin waves, and cytokinesis. In each case, we compare and contrast various published models, highlight the relevant aspects of the biology, and provide a sense of the direction in which the field is moving.

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.

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.

Structural analyses of protein–protein interactions are required to reveal their functional mechanisms, and accurate protein–protein complex models, based on experimental results, are the starting points for drug development. In addition, structural information about proteins under physiologically relevant conditions is crucially important for understanding biological events. However, for proteins such as those embedded in lipid bilayers and transiently complexed with their effectors under physiological conditions, structural analyses by conventional methods are generally difficult, due to their large molecular weights and inhomogeneity. We have developed the cross-saturation (CS) method, which is an nuclear magnetic resonance measurement technique for the precise identification of the interfaces of protein–protein complexes. In addition, we have developed an extended version of the CS method, termed transferred cross-saturation (TCS), which enables the identification of the residues of protein ligands in close proximity to huge (>150 kDa) and heterogeneous complexes under fast exchange conditions (>0.1 s−1). Here, we discuss the outline, basic theory, and practical considerations of the CS and TCS methods. In addition, we will review the recent progress in the construction of models of protein–protein complexes, based on CS and TCS experiments, and applications of TCS to in situ analyses of biologically and medically important proteins in physiologically relevant states.