We review what has been learned about the protein-folding problem from experimental kinetic studies. These studies reveal patterns of both great richness and surprising simplicity. The patterns can be interpreted in terms of proteins possessing an energy landscape which is largely, but not completely, funnel-like. Issues such as speed limitations of folding, the robustness of folding, the origin of barriers and cooperativity and the ensemble nature of transition states, intermediate and traps are assessed using the results from several experimental groups highlighting energy-landscape ideas as an interpretive framework.
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.
Using atomistic simulations, we show the formation of stable triplet structure when particular GC-rich DNA duplexes are extended in solution over a timescale of hundreds of nanoseconds, in the presence of organic salt. We present planar-stacked triplet disproportionated DNA (Σ DNA) as a possible solution phase of the double helix under tension, subject to sequence and the presence of stabilising co-factors. Considering the partitioning of the duplexes into triplets of base pairs as the first step of operation of recombinase enzymes like RecA, we emphasise the structure-function relationship in Σ DNA. We supplement atomistic calculations with thermodynamic arguments to show that codons for 'phase 1' amino acids (those appearing early in evolution) are more likely than a lower entropy GC-rich sequence to form triplets under tension. We further observe that the four amino acids supposed (in the 'GADV world' hypothesis) to constitute the minimal set to produce functional globular proteins have the strongest triplet-forming propensity within the phase 1 set, showing a series of decreasing triplet propensity with evolutionary newness. The weak form of our observation provides a physical mechanism to minimise read frame and recombination alignment errors in the early evolution of the genetic code.
In this review we propose to address the question: for the life-science researcher, what does X-ray microscopy have to offer that is not otherwise easily available?We will see that the answer depends on a combination of resolution, penetrating power, analytical sensitivity, compatibility with wet specimens, and the ease of image interpretation.
Among the Various methods for characterizing macromolecules in solution, hydrodynamic techniques play a major role. Since the advent of the ultracentrifuge and the development of viscometric apparatus, sedimentation coefficients and intrinsic viscosities have been extensively used to learn about the size and shape of synthetic and biological polymers. More recently, refined techniques such as quasielastic light scattering, transient electric birefringence and fluorescence anisotropy decay have made it possible to obtain in a simple and rapid way quantitative information of high precision on the translational and rotational brownian dynamics of dissolved macromolecules.
An exciting aspect of NMR spectroscopy is its ability to monitor, non-invasively, a variety of small molecules in cells and tissues. This leads to the possibility of investigating details of cellular biochemistry previously obscured by separation and purification procedures.
Proton and carbon-13 nmr spectra of unsonicated lipid bilayers and biological membranes are generally dominated by strong proton–proton and proton–carbon dipolar interactions. As a result the spectra contain a large number of overlapping resonances and are rather difficult to analyse. Nevertheless, important information on the structure and dynamic behaviour of lipid systems has been provided by these techniques (Wennerström & Lindblom, 1977).
The scientific community will remember Peter Läuger as an exceptional man combining a generous personality and a sharp and skilful mind. He was able to attract by his views the interest of a large spectrum of biologists concerned by the mechanism of ion translocation through membranes. Yet, he was not a man with a single technique or theory. Using an authentically multidisciplinary approach, his ambition was to ‘understand transmembrane transport at the microscopic level, to capture its dynamics in the course of defined physiological processes’ (1987). According to him, ‘new concepts in the molecular physics of proteins’ had to be imagined, and ‘the traditional static picture of proteins has been replaced by the notions that proteins represent dynamic structures, subjected to conformational fluctuations covering a very wide time-range’ (1987).
Exosomes are bioactive vesicles released from multivesicular bodies by intact cells and participate in intercellular signalling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a Fatty Acid Binding Protein, the exosomes contained the whole set of phospholipases (A2, C and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the PLD / PAP1 pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes, i.e. the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPgammaS triggered activation of PLA2s and PLD2. A large panel of free fatty acids, including arachidonic acid, and derivatives such as prostaglandin E2 and 15-deoxy-Delta12,14-prostaglandinJ2 were detected. We observed that the exosomes were internalized by resting and activated RBLcells, and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins, i.e. concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.
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.
Crystallography supplies unparalleled detail on structural information critical for mechanistic analyses; however, it is restricted to describing low energy conformations of macromolecules within crystal lattices. Small angle X-ray scattering (SAXS) offers complementary information about macromolecular folding, unfolding, aggregation, extended conformations, flexibly linked domains, shape, conformation, and assembly state in solution, albeit at the lower resolution range of about 50 A to 10 A resolution, but without the size limitations inherent in NMR and electron microscopy studies. Together these techniques can allow multi-scale modeling to create complete and accurate images of macromolecules for modeling allosteric mechanisms, supramolecular complexes, and dynamic molecular machines acting in diverse processes ranging from eukaryotic DNA replication, recombination and repair to microbial membrane secretion and assembly systems. This review addresses both theoretical and practical concepts, concerns and considerations for using these techniques in conjunction with computational methods to productively combine solution scattering data with high-resolution structures. Detailed aspects of SAXS experimental results are considered with a focus on data interpretation tools suitable to model protein and nucleic acid macromolecular structures, including membrane protein, RNA, DNA, and protein-nucleic acid complexes. The methods discussed provide the basis to examine molecular interactions in solution and to study macromolecular flexibility and conformational changes that have become increasingly relevant for accurate understanding, simulation, and prediction of mechanisms in structural cell biology and nanotechnology.
Phosphoryl transfer plays key roles in signaling, energy transduction, protein synthesis, and maintaining the integrity of the genetic material. On the surface, it would appear to be a simple nucleophile displacement reaction. However, this simplicity is deceptive, as, even in aqueous solution, the low-lying d-orbitals on the phosphorus atom allow for eight distinct mechanistic possibilities, before even introducing the complexities of the enzyme catalyzed reactions. To further complicate matters, while powerful, traditional experimental techniques such as the use of linear free-energy relationships (LFER) or measuring isotope effects cannot make unique distinctions between different potential mechanisms. A quarter of a century has passed since Westheimer wrote his seminal review, 'Why Nature Chose Phosphate' (Science 235 (1987), 1173), and a lot has changed in the field since then. The present review revisits this biologically crucial issue, exploring both relevant enzymatic systems as well as the corresponding chemistry in aqueous solution, and demonstrating that the only way key questions in this field are likely to be resolved is through careful theoretical studies (which of course should be able to reproduce all relevant experimental data). Finally, we demonstrate that the reason that nature really chose phosphate is due to interplay between two counteracting effects : on the one hand, phosphates are negatively charged and the resulting charge-charge repulsion with the attacking nucleophile contributes to the very high barrier for hydrolysis, making phosphate esters among the most inert compounds known. However, biology is not only about reducing the barrier to unfavorable chemical reactions. That is, the same charge-charge repulsion that makes phosphate ester hydrolysis so unfavorable also makes it possible to regulate, by exploiting the electrostatics. This means that phosphate ester hydrolysis can not only be turned on, but also be turned off, by fine tuning the electrostatic environment and the present review demonstrates numerous examples where this is the case. Without this capacity for regulation, it would be impossible to have for instance a signaling or metabolic cascade, where the action of each participant is determined by the fine-tuned activity of the previous piece in the production line. This makes phosphate esters the ideal compounds to facilitate life as we know it.
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
Nucleases cleave the phosphodiester bonds of nucleic acids and may be endo or exo, DNase or RNase, topoisomerases, recombinases, ribozymes, or RNA splicing enzymes. In this review, 1 survey nuclease activities with known structures and catalytic machinery and classify them by reaction mechanism and metal-ion dependence and by their biological function ranging from DNA replication, recombination, repair, RNA maturation, processing, interference, to defense, nutrient regeneration or cell death. Several general principles emerge from this analysis. There is little correlation between catalytic mechanism and biological function. A single catalytic mechanism can be adapted in a variety of reactions and biological pathways. Conversely, a single biological process can often be accomplished by multiple tertiary and quaternary folds and by more than one catalytic mechanism. Two-metal-ion-dependent nucleases comprise the largest number of different tertiary folds and mediate the most diverse set of biological functions. Metal-ion-dependent cleavage is exclusively associated with exonucleases producing mononucleotides and endonucleases that cleave double- or single-stranded substrates in helical and base-stacked conformations. All metal-ion-independent RNases generate 2',3'-cyclic phosphate products, and all metal-ion-independent DNases form phospho-protein intermediates. I also find several previously unnoted relationships between different nucleases and shared catalytic configurations.
There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2-to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.
Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT3, GABA(A) and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT3) and some are anion selective (e.g. GABA(A) and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience. In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them.
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.