Showing papers in "Annual Review of Biophysics in 2010"
TL;DR: The determinants and functional implications of the subcellular distribution and membrane topology of the most abundant negatively charged phospholipid in eukaryotic membranes are discussed.
Abstract: Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in eukaryotic membranes. PS directs the binding of proteins that bear C2 or gamma-carboxyglutamic domains and contributes to the electrostatic association of polycationic ligands with cellular membranes. Rather than being evenly distributed, PS is found preferentially in the inner leaflet of the plasma membrane and in endocytic membranes. The loss of PS asymmetry is an early indicator of apoptosis and serves as a signal to initiate blood clotting. This review discusses the determinants and functional implications of the subcellular distribution and membrane topology of PS.
793 citations
TL;DR: Observed correlations between functional changes in structures observed in experiments and the global motions predicted by coarse-grained analyses suggest that computational methods may be advantageously employed for assessingfunctional changes in structure and allosteric mechanisms intrinsically favored by the native fold.
Abstract: Biomolecular systems possess unique, structure-encoded dynamic properties that underlie their biological functions. Recent studies indicate that these dynamic properties are determined to a large extent by the topology of native contacts. In recent years, elastic network models used in conjunction with normal mode analyses have proven to be useful for elucidating the collective dynamics intrinsically accessible under native state conditions, including in particular the global modes of motions that are robustly defined by the overall architecture. With increasing availability of structural data for well-studied proteins in different forms (liganded, complexed, or free), there is increasing evidence in support of the correspondence between functional changes in structures observed in experiments and the global motions predicted by these coarse-grained analyses. These observed correlations suggest that computational methods may be advantageously employed for assessing functional changes in structure and allosteric mechanisms intrinsically favored by the native fold.
550 citations
TL;DR: By exploiting the genetic advantages of Dictyostelium, investigators are working out the complex network of interactions between the proteins that have been implicated in the chemotactic processes of motility, directional sensing, and polarity.
Abstract: Chemotaxis, the directed migration of cells in chemical gradients, is a vital process in normal physiology and in the pathogenesis of many diseases. Chemotactic cells display motility, directional sensing, and polarity. Motility refers to the random extension of pseudopodia, which may be driven by spontaneous actin waves that propagate through the cytoskeleton. Directional sensing is mediated by a system that detects temporal and spatial stimuli and biases motility toward the gradient. Polarity gives cells morphologically and functionally distinct leading and lagging edges by relocating proteins or their activities selectively to the poles. By exploiting the genetic advantages of Dictyostelium, investigators are working out the complex network of interactions between the proteins that have been implicated in the chemotactic processes of motility, directional sensing, and polarity.
476 citations
TL;DR: This review describes the recent progress in understanding the structural rearrangements triggered by actin binding that are coupled to force generation and product release and describes studies that delineate how some classes of myosin motors are adapted for processive movement on actin.
Abstract: The general structural features of the motor region of myosin superfamily members are now well established, as is a subset of the structural and kinetic transitions of the actin-myosin catalytic cycle. Not yet visualized are the structural rearrangements triggered by actin binding that are coupled to force generation and product release. In this review we describe the recent progress in understanding these missing components of the mechanism of chemomechanical transduction by myosin motors. These insights come from a combination of kinetic and single-molecule studies on multiple classes of myosins, with additional insights from contracting muscle fibers. These recent studies have explored the effects of intermediate and high loads on the kinetics of the actin-bound myosin state transitions. We also describe studies that delineate how some classes of myosin motors are adapted for processive movement on actin.
356 citations
TL;DR: Novel biophysical methods involving single-molecule measurements should foster progress in the detailed molecular mechanisms of ATP hydrolysis/Pi release on F-actin remain elusive, as well as the mechanism of filament branching with Arp2/3 complex or that of the formin-driven processive actin assembly.
Abstract: Recent advances in structural, biochemical, biophysical, and live cell imaging approaches have furthered our understanding of the molecular mechanisms by which regulated assembly dynamics of actin filaments drive motile processes. Attention is focused on lamellipodium protrusion, powered by the turnover of a branched filament array. ATP hydrolysis on actin is the key reaction that allows filament treadmilling. It regulates barbed-end dynamics and length fluctuations at steady state and specifies the functional interaction of actin with essential regulatory proteins such as profilin and ADF/cofilin. ATP hydrolysis on actin and Arp2/3 acts as a timer, regulating the assembly and disassembly of the branched array to generate tropomyosin-mediated heterogeneity in the structure and dynamics of the lamellipodial network. The detailed molecular mechanisms of ATP hydrolysis/Pi release on F-actin remain elusive, as well as the mechanism of filament branching with Arp2/3 complex or that of the formin-driven processive actin assembly. Novel biophysical methods involving single-molecule measurements should foster progress in these crucial issues.
335 citations
TL;DR: A range of established and new strategies that used to be reserved for metalloproteins is becoming available for NMR spectroscopy of nonmetalloproteinins with new reagents for site-specific labeling of proteins with paramagnetic metal ions.
Abstract: Paramagnetic metal ions offer outstanding opportunities for protein studies by nuclear magnetic resonance (NMR) spectroscopy. The paramagnetic effects manifested in the NMR spectra provide powerful restraints for the determination of the three-dimensional structure of proteins, open new possibilities for the analysis of protein-protein and protein-ligand interactions, and offer widely applicable tools for sensitivity enhancement of NMR experiments, resonance assignments, and studies of conformational heterogeneity and exchange. With the advent of new reagents for site-specific labeling of proteins with paramagnetic metal ions, a range of established and new strategies that used to be reserved for metalloproteins is becoming available for NMR spectroscopy of nonmetalloproteins.
331 citations
TL;DR: Recent work has suggested or confirmed that diverse types of channels, including TRP channels, K(2P) channels, MscS-like proteins, and DEG/ENaC channels, are mechanically gated.
Abstract: Mechanosensitive ion channels are gated directly by physical stimuli and transduce these stimuli into electrical signals. Several criteria must apply for a channel to be considered mechanically gated. Mechanosensitive channels from bacterial systems have met these criteria, but few eukaryotic channels have been confirmed by the same standards. Recent work has suggested or confirmed that diverse types of channels, including TRP channels, K2P channels, MscS-like proteins, and DEG/ENaC channels, are mechanically gated. Several studies point to the importance of the plasma membrane for channel gating, but intracellular and/or extracellular structures may also be required.
329 citations
TL;DR: Recent work that uses synthetic biology approaches to investigate the organization and function of cellular networks are outlined, and a vision for a synthetic biology toolkit that could be used to interrogate the design principles of diverse systems is described.
Abstract: The living cell is an incredibly complex entity, and the goal of predictively and quantitatively understanding its function is one of the next great challenges in biology. Much of what we know about the cell concerns its constituent parts, but to a great extent we have yet to decode how these parts are organized to yield complex physiological function. Classically, we have learned about the organization of cellular networks by disrupting them through genetic or chemical means. The emerging discipline of synthetic biology offers an additional, powerful approach to study systems. By rearranging the parts that comprise existing networks, we can gain valuable insight into the hierarchical logic of the networks and identify the modular building blocks that evolution uses to generate innovative function. In addition, by building minimal toy networks, one can systematically explore the relationship between network structure and function. Here, we outline recent work that uses synthetic biology approaches to investigate the organization and function of cellular networks, and describe a vision for a synthetic biology toolkit that could be used to interrogate the design principles of diverse systems.
195 citations
TL;DR: Linkages between theoretical and experimental studies of phase separation in lipid bilayer model membranes are summarized to summarizes whether lipid nanodomains can exist in stable equilibrium in membranes and what is the distribution of their sizes and lifetimes in membranes of different composition.
Abstract: Lipid bilayer model membranes that contain a single lipid species can undergo transitions between ordered and disordered phases, and membranes that contain a mixture of lipid species can undergo phase separations. Studies of these transformations are of interest for what they can tell us about the interaction energies of lipid molecules of different species and conformations. Nanoscopic phases (<200 nm) can provide a model for membrane rafts, specialized membrane domains enriched in cholesterol and sphingomyelin, which are believed to have essential biological functions in cell membranes. Crucial questions are whether lipid nanodomains can exist in stable equilibrium in membranes and what is the distribution of their sizes and lifetimes in membranes of different composition. Theoretical methods have supplied much information on these questions, but better experimental methods are needed to detect and characterize nanodomains under normal membrane conditions. This review summarizes linkages between theoretical and experimental studies of phase separation in lipid bilayer model membranes.
193 citations
TL;DR: In this article, a coarse-grained molecular transfer model was proposed to predict the intricate details of protein folding as a function of denaturant concentration, which can be used to solve the protein folding problem in the broadest sense.
Abstract: Understanding how monomeric proteins fold under in vitro conditions is crucial to describing their functions in the cellular context. Significant advances in theory and experiments have resulted in a conceptual framework for describing the folding mechanisms of globular proteins. The sizes of proteins in the denatured and folded states, cooperativity of the folding transition, dispersions in the melting temperatures at the residue level, and timescales of folding are, to a large extent, determined by N, the number of residues. The intricate details of folding as a function of denaturant concentration can be predicted by using a novel coarse-grained molecular transfer model. By watching one molecule fold at a time, using single-molecule methods, investigators have established the validity of the theoretically anticipated heterogeneity in the folding routes and the N-dependent timescales for the three stages in the approach to the native state. Despite the successes of theory, of which only a few examples are documented here, we conclude that much remains to be done to solve the protein folding problem in the broadest sense.
190 citations
TL;DR: O Ongoing development of microfluidic techniques and their integration with protein production, characterization, and in situ diffraction promises to accelerate the progress of structural biology.
Abstract: To obtain protein crystals, researchers must search for conditions in multidimensional chemical space. Empirically, thousands of crystallization experiments are carried out to screen various precipitants at multiple concentrations. Microfluidics can manipulate fluids on a nanoliter scale, and it affects crystallization twofold. First, it miniaturizes the experiments that can currently be done on a larger scale and enables crystallization of proteins that are available only in small amounts. Second, it offers unique experimental approaches that are difficult or impossible to implement on a larger scale. Ongoing development of microfluidic techniques and their integration with protein production, characterization, and in situ diffraction promises to accelerate the progress of structural biology.
TL;DR: Current structural, evolutionary, and mechanistic ideas are discussed, along with genomic studies for exploring the function and diversity of this family of bacterial organelles.
Abstract: Some bacteria contain organelles or microcompartments consisting of a large virion-like protein shell encapsulating sequentially acting enzymes. These organized microcompartments serve to enhance or protect key metabolic pathways inside the cell. The variety of bacterial microcompartments provide diverse metabolic functions, ranging from CO2 fixation to the degradation of small organic molecules. Yet they share an evolutionarily related shell, which is defined by a conserved protein domain that is widely distributed across the bacterial kingdom. Structural studies on a number of these bacterial microcompartment shell proteins are illuminating the architecture of the shell and highlighting its critical role in controlling molecular transport into and out of microcompartments. Current structural, evolutionary, and mechanistic ideas are discussed, along with genomic studies for exploring the function and diversity of this family of bacterial organelles.
TL;DR: The emerging model of activation indicates that, rather than being unique, the visual receptors provide a basis for understanding the common structural and dynamic elements in the class A GPCRs.
Abstract: Rhodopsin is a specialized G protein-coupled receptor (GPCR) found in vertebrate rod cells. Absorption of light by its 11-cis retinal chromophore leads to rapid photochemical isomerization and receptor activation. Recent results from protein crystallography and NMR spectroscopy show how structural changes on the extracellular side of rhodopsin induced by retinal isomerization are coupled to the motion of membrane-spanning helices to create a G protein binding pocket on the intracellular side of the receptor. The signaling pathway provides a comprehensive explanation for the conservation of specific amino acids and structural motifs across the class A family of GPCRs, as well as for the conservation of selected residues within the visual receptor subfamily. The emerging model of activation indicates that, rather than being unique, the visual receptors provide a basis for understanding the common structural and dynamic elements in the class A GPCRs.
TL;DR: By coupling helix assembly with nascent tertiary interactions, compact folding intermediates in RNA also play a crucial role in ligand binding and RNA-protein recognition.
Abstract: Large noncoding RNAs fold into their biologically functional structures via compact yet disordered intermediates, which couple the stable secondary structure of the RNA with the emerging tertiary fold. The specificity of the collapse transition, which coincides with the assembly of helical domains, depends on RNA sequence and counterions. It determines the specificity of the folding pathways and the magnitude of the free energy barriers to the ensuing search for the native conformation. By coupling helix assembly with nascent tertiary interactions, compact folding intermediates in RNA also play a crucial role in ligand binding and RNA-protein recognition.
TL;DR: A partitioning model of the Golgi apparatus is discussed as a working hypothesis to explain how membrane lipids and proteins that are segregated based on lateral lipid partitioning support the unique composition of the biosynthetic and endocytic recycling pathways in the face of constant trafficking of molecular constituents.
Abstract: The endomembrane system of eukaryotic cells uses membrane-enclosed carriers to move diverse macromolecules among different membrane-bound compartments, a requirement for cells to secrete and take up molecules from their environment. Two recycling pathways—biosynthetic and endocytic, each with specific lipid components—make up this system, with the Golgi apparatus mediating transport between the two. Here, we integrate lipid-based mechanisms into the description of this system. A partitioning model of the Golgi apparatus is discussed as a working hypothesis to explain how membrane lipids and proteins that are segregated based on lateral lipid partitioning support the unique composition of the biosynthetic and endocytic recycling pathways in the face of constant trafficking of molecular constituents. We further discuss how computational modeling can allow for interpretation of experimental findings and provide mechanistic insight into these important cellular pathways.
TL;DR: Current experiments with implications for the origin of life are reviewed, emphasizing the ability of unexpected physical processes to facilitate the self-assembly and self-replication of the first biological systems.
Abstract: Recent synthetic approaches to understanding the origin of life have yielded insights into plausible pathways for the emergence of the first cells. Here we review current experiments with implications for the origin of life, emphasizing the ability of unexpected physical processes to facilitate the self-assembly and self-replication of the first biological systems. These laboratory efforts have uncovered novel physical mechanisms for the emergence of homochirality; the concentration and purification of prebiotic building blocks; and the ability of the first cells to assemble, grow, divide, and acquire greater complexity. In the absence of evolved biochemical capabilities, such physical processes likely played an essential role in early biology.
TL;DR: This review describes photostimulation methods, their applications, and opportunities for further advancement, including photorelease of caged neurotransmitters, engineered light-gated receptors and channels, and naturally light-sensitive ion channels and pumps.
Abstract: Advances in optics, genetics, and chemistry have enabled the investigation of brain function at all levels, from intracellular signals to single synapses, whole cells, circuits, and behavior. Until recent years, these research tools have been utilized in an observational capacity: imaging neural activity with fluorescent reporters, for example, or correlating aberrant neural activity with loss-of-function and gain-of-function pharmacological or genetic manipulations. However, optics, genetics, and chemistry have now combined to yield a new strategy: using light to drive and halt neuronal activity with molecular specificity and millisecond precision. Photostimulation of neurons is noninvasive, has unmatched spatial and temporal resolution, and can be targeted to specific classes of neurons. The optical methods developed to date encompass a broad array of strategies, including photorelease of caged neurotransmitters, engineered light-gated receptors and channels, and naturally light-sensitive ion channels and pumps. In this review, we describe photostimulation methods, their applications, and opportunities for further advancement.
TL;DR: The most recent structural models of the bacterial Ribosome that shed light on the movement of messenger RNA and transfer RNA on the ribosome after each peptide bond is formed are described, a process termed translocation.
Abstract: Protein biosynthesis, or translation, occurs on the ribosome, a large RNA-protein assembly universally conserved in all forms of life. Over the last decade, structures of the small and large ribosomal subunits and of the intact ribosome have begun to reveal the molecular details of how the ribosome works. Both cryo-electron microscopy and X-ray crystallography continue to provide fresh insights into the mechanism of translation. In this review, we describe the most recent structural models of the bacterial ribosome that shed light on the movement of messenger RNA and transfer RNA on the ribosome after each peptide bond is formed, a process termed translocation. We also discuss recent structures that reveal the molecular basis for stop codon recognition during translation termination. Finally, we review recent advances in understanding how bacteria handle errors in both translocation and termination.
TL;DR: This review discusses the achievements and promise of a bottom-up approach that uses well-characterized subnetworks as model systems for understanding larger networks and highlights the potential of negative and positive feedback, as well as their combinations, to generate robust homeostasis, epigenetics, and oscillations.
Abstract: The function of living cells is controlled by complex regulatory networks that are built of a wide diversity of interacting molecular components. The sheer size and intricacy of molecular networks of even the simplest organisms are obstacles toward understanding network functionality. This review discusses the achievements and promise of a bottom-up approach that uses well-characterized subnetworks as model systems for understanding larger networks. It highlights the interplay between the structure, logic, and function of various types of small regulatory circuits. The bottom-up approach advocates understanding regulatory networks as a collection of entangled motifs. We therefore emphasize the potential of negative and positive feedback, as well as their combinations, to generate robust homeostasis, epigenetics, and oscillations.
TL;DR: The contributions of single-molecule studies to the understanding of the dynamic nature of translation are covered, including real-time observation of ribosome movement and dynamics during translation.
Abstract: Our current understanding of the mechanism of translation is based on nearly fifty years of biochemical and biophysical studies. This mechanism, which requires the ribosome to manipulate tRNA and step repetitively along the mRNA, implies movement. High-resolution structures of the ribosome and its ligands have recently described translation in atomic detail, capturing the endpoints of large-scale rearrangements of the ribosome. Direct observation of the dynamic events that underlie the mechanism of translation is challenged by ensemble averaging in bulk solutions. Single-molecule methods, which eliminate these averaging effects, have emerged as powerful tools to probe the mechanism of translation. Single-molecule fluorescence experiments have described the dynamic motion of the ribosome and tRNA. Single-molecule force measurements have directly probed the forces stabilizing ribosomal complexes. Recent developments have allowed real-time observation of ribosome movement and dynamics during translation. This review covers the contributions of single-molecule studies to our understanding of the dynamic nature of translation.
TL;DR: How mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes is discussed, which includes the rate ofActin assembly at the leading edge of a moving cell, the disassembly rates of intracellular actin networks, the polymerization time course in externally stimulated cells, and spontaneous spatiotemporal patterns formed by actin.
Abstract: The dynamic nature of actin in cells manifests itself constantly. Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of actin at the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The dynamic properties addressed include the rate of actin assembly at the leading edge of a moving cell, the disassembly rates of intracellular actin networks, the polymerization time course in externally stimulated cells, and spontaneous spatiotemporal patterns formed by actin. Although several aspects of actin assembly have been clarified by increasingly sophisticated models, our understanding of rapid actin disassembly is limited, and the origins of nonmonotonic features in externally stimulated actin polymerization remain unclear. Theory has generated several concrete, testable hypotheses for the origins of spontaneous actin waves and cell-edge oscillations. The development and use of more biomimetic systems applicable to the geometry of a cell will be key to obtaining a quantitative understanding of actin dynamics in cells.
TL;DR: This work reports on recent progress in the biophysics of knotting-the formation, characterization, and dynamics of knots in various biophysical contexts.
Abstract: Knots appear in a wide variety of biophysical systems, ranging from biopolymers, such as DNA and proteins, to macroscopic objects, such as umbilical cords and catheters. Although significant advancements have been made in the mathematical theory of knots and some progress has been made in the statistical mechanics of knots in idealized chains, the mechanisms and dynamics of knotting in biophysical systems remain far from fully understood. We report on recent progress in the biophysics of knotting—the formation, characterization, and dynamics of knots in various biophysical contexts.
TL;DR: Single-molecule methods that allow the study of individual replication proteins and their functioning within the replisome are described, leading to an improved understanding of the molecular mechanisms underlying DNA replication.
Abstract: Replication of DNA is carried out by the replisome, a multiprotein complex responsible for the unwinding of parental DNA and the synthesis of DNA on each of the two DNA strands. The impressive speed and processivity with which the replisome duplicates DNA are a result of a set of tightly regulated interactions between the replication proteins. The transient nature of these protein interactions makes it challenging to study the dynamics of the replisome by ensemble-averaging techniques. This review describes single-molecule methods that allow the study of individual replication proteins and their functioning within the replisome. The ability to mechanically manipulate individual DNA molecules and record the dynamic behavior of the replisome while it duplicates DNA has led to an improved understanding of the molecular mechanisms underlying DNA replication.
TL;DR: The combined enzymatic and voltage control of a DNA molecule in the nanopore can be used to sequence the DNA.
Abstract: When a voltage is imposed across a thin membrane containing a nanoscopic pore, the electric field generated within the pore captures linear ionized polymers, such as nucleic acids, that are present in the solution bathing the pore. The nucleic acid molecule transiently blocks ionic current as it is translocated through the pore, and modulations of the current provide information about the structure and dynamic motion of the molecule. Altering the imposed voltage allows movement of the DNA molecule in the pore to be controlled. If a DNA-processing enzyme such as an exonuclease or polymerase is present, the enzyme-DNA complex is also drawn to the pore, and further modulations of the ionic current reflect enzyme function at the single-molecule level on millisecond timescales. The combined enzymatic and voltage control of a DNA molecule in the nanopore can be used to sequence the DNA.
TL;DR: Pi, Pi, and Pi reveal several salient points that aid the understanding of mechanochemical coupling that are important for analyzing hexameric helicase, F(1)F(0) ATPase, and kinesin.
Abstract: How do molecular motors convert chemical energy to mechanical work? Helicases and nucleic acids offer simple motor systems for extensive biochemical and biophysical analyses. Atomic resolution structures of UvrD-like helicases complexed with DNA in the presence of AMPPNP, ADP.Pi, and Pi reveal several salient points that aid our understanding of mechanochemical coupling. Each ATPase cycle causes two motor domains to rotationally close and open. At a minimum, two motor-track contact points of alternating tight and loose attachment convert domain rotations to unidirectional movement. A motor is poised for action only when fully in contact with its track and, if applicable, working against a load. The orientation of domain rotation relative to the track determines whether the movement is linear, spiral, or circular. Motors powered by ATPases likely deliver each power stroke in two parts, before and after ATP hydrolysis. Implications of these findings for analyzing hexameric helicase, F(1)F(0) ATPase, and kinesin are discussed.
TL;DR: The theory and practice of heavy isotope internal standards for isolating and identifying experimental variables such as extraction or fractionation efficiencies are discussed and a review of significant recent biological applications is provided.
Abstract: Genetic, chemical, and environmental perturbations can all induce large changes in cellular proteomes, and research aimed at quantifying these changes are an important part of modern biology. Although improvements in the hardware and software of mass spectrometers have produced increased throughput and accuracy of such measurements, new uses of heavy isotope internal standards that assist in this process have emerged. Surprisingly, even complex life forms such as mammals can be grown to near-complete replacement with heavy isotopes of common biological elements such as 15N, and these isotopically labeled organisms provide excellent controls for isolating and identifying experimental variables such as extraction or fractionation efficiencies. We discuss here the theory and practice of these technologies, as well as provide a review of significant recent biological applications.
TL;DR: This research was curiosity driven as well as problem and technique oriented and showed that antigenic peptides bind to reconstituted class II MHC molecules in membranes and trigger specific T-helper cells.
Abstract: My research has included chemical physics, electron and NMR spectroscopy, membrane biophysics, and immunology. This research was curiosity driven as well as problem and technique oriented. A theoretical equation was developed for relating nuclear hyperfine splittings to electron spin distributions in free radicals. Another equation was developed to relate NMR spectra to chemical reaction rates. Early evidence for the liquid-like properties of cell membranes was obtained through the use of paramagnetic probes (spin labels). Spin labels were used in measurements of lateral as well as transverse diffusion of phospholipids in bilayer membranes. Liquid-liquid phase separations were discovered in monolayer membranes containing phospholipids and cholesterol. In the area of immunology, it was shown that antigenic peptides bind to reconstituted class II MHC molecules in membranes and trigger specific T-helper cells.