Abstract: 2007 Publication year: 2007 Source:Advances in Protein Chemistry, Volume 74
This chapter addresses, from a molecular structural perspective gained from examination of x‐ray crystallographic and biochemical data, the mechanisms by which GTP‐bound Gα subunits of heterotrimeric G proteins recognize and regulate effectors. The mechanism of GTP hydrolysis by Gα and rate acceleration by GAPs are also considered. The effector recognition site in all Gα homologues is formed almost entirely of the residues extending from the C‐terminal half of α2 (Switch II) together with the α3 helix and its junction with the β5 strand. Effector binding does not induce substantial changes in the structure of Gα•GTP. Effectors are structurally diverse. Different effectors may recognize distinct subsets of effector‐binding residues of the same Gα protein. Specificity may also be conferred by differences in the main chain conformation of effector‐binding regions of Gα subunits. Several Gα regulatory mechanisms are operative. In the regulation of GMP phospodiesterase, Gαt sequesters an inhibitory subunit. Gαs is an allosteric activator and inhibitor of adenylyl cyclase, and Gαi is an allosteric inhibitor. Gαq does not appear to regulate GRK, but is rather sequestered by it. GTP hydrolysis terminates the signaling state of Gα. The binding energy of GTP that is used to stabilize the Gα:effector complex is dissipated in this reaction. Chemical steps of GTP hydrolysis, specifically, formation of a dissociative transition state, is rate limiting in Ras, a model G protein GTPase, even in the presence of a GAP; however, the energy of enzyme reorganization to produce a catalytically active conformation appears to be substantial. It is possible that the collapse of the switch regions, associated with Gα deactivation, also encounters a kinetic barrier, and is coupled to product (Pi) release or an event preceding formation of the GDP•Pi complex. Evidence for a catalytic intermediate, possibly metaphosphate, is discussed. Gα GAPs, whether exogenous proteins or effector‐linked domains, bind to a discrete locus of Gα that is composed of Switch I and the N‐terminus of Switch II. This site is immediately adjacent to, but does not substantially overlap, the Gα effector binding site. Interactions of effectors and exogenous GAPs with Gα proteins can be synergistic or antagonistic, mediated by allosteric interactions among the three molecules. Unlike GAPs for small GTPases, Gα GAPs supply no catalytic residues, but rather appear to reduce the activation energy for catalytic activation of the Gα catalytic site.
Abstract: 2007 Publication year: 2007 Source:Advances in Protein Chemistry, Volume 74
Heterotrimeric G proteins couple the activation of heptahelical receptors at the cell surface to the intracellular signaling cascades that mediate the physiological responses to extracellular stimuli. G proteins are molecular switches that are activated by receptor‐catalyzed GTP for GDP exchange on the G protein α subunit, which is the rate‐limiting step in the activation of all downstream signaling. Despite the important biological role of the receptor–G protein interaction, relatively little is known about the structure of the complex and how it leads to nucleotide exchange. This chapter will describe what is known about receptor and G protein structure and outline a strategy for assembling the current data into improved models for the receptor–G protein complex that will hopefully answer the question as to how receptors flip the G protein switch.
Abstract: 2007 Publication year: 2007 Source:Advances in Protein Chemistry, Volume 74
The binding of full and partial agonist ligands (L) to G protein–coupled receptors (GPCRs) initiates the formation of ternary complexes with G proteins [ligand–receptor‐G protein (LRG) complexes]. Cyclic ternary complex models are required to account for the thermodynamically plausible complexes. It has recently become possible to assemble solubilized formyl peptide receptor (FPR) and β2‐adrenergic receptor (β2AR) ternary complexes for flow cytometric bead‐based assays. In these systems, soluble ternary complex formation of the receptors with G proteins allows direct quantitative measurements which can be analyzed in terms of three‐dimensional concentrations (molarity). In contrast to the difficulty of analyzing comparable measurements in two‐dimensional membrane systems, the output of these flow cytometric experiments can be analyzed via ternary complex simulations in which all of the parameters can be estimated. An outcome from such analysis yielded lower affinity for soluble ternary complex assembly by partial agonists compared with full agonists for the β2AR. In the four‐sided ternary complex model, this behavior is consistent with distinct ligand‐induced conformational states for full and partial agonists. Rapid mix flow cytometry is used to analyze the subsecond dynamics of guanine nucleotide‐mediated ternary complex disassembly. The modular breakup of ternary complex components is highlighted by the finding that the fastest step involves the departure of the ligand‐activated GPCR from the intact G protein heterotrimer. The data also show that, under these experimental conditions, G protein subunit dissociation does not occur within the time frame relevant to signaling. The data and concepts are discussed in the context of a review of current literature on signaling mechanism based on structural and spectroscopic (FRET) studies of ternary complex components.
Abstract: 2007 Publication year: 2007 Source:Advances in Protein Chemistry, Volume 74
G protein–coupled receptors (GPCRs) mediate responses to hormones and neurotransmitters, as well as the senses of sight, smell, and taste. These remarkably versatile signaling molecules respond to structurally diverse ligands. Many GPCRs couple to multiple G protein subtypes, and several have been shown to activate G protein–independent signaling pathways. Drugs acting on GPCRs exhibit efficacy profiles that may differ for different signaling cascades. The functional plasticity exhibited by GPCRs can be attributed to structural flexibility and the existence of multiple ligand‐specific conformational states. This chapter will review our current understanding of the mechanism by which agonists bind and activate GPCRs.
Abstract: 2007 Publication year: 2007 Source:Advances in Protein Chemistry, Volume 74
We describe and review methods for the kinetic analysis of G protein–coupled receptor (GPCR) activation and signaling that are based on optical methods. In particular, we describe the use of fluorescence resonance energy transfer (FRET) as a means of analyzing conformational changes within a single protein (for example a receptor) or between subunits of a protein complex (such as a G protein heterotrimer) and finally between distinct proteins (such as a receptor and a G protein). These methods allow the analysis of signaling kinetics in intact cells with proteins that retain their essential functional properties. They have produced a number of unexpected results: fast receptor activation kinetics in the millisecond range, similarly fast kinetics for receptor–G protein interactions, but much slower activation kinetics for G protein activation.
Abstract: 2007 Publication year: 2007 Source:Advances in Protein Chemistry, Volume 74
Monomeric Rho GTPases regulate cellular dynamics through remodeling of the cytoskeleton, modulation of immediate signaling pathways, and longer‐term regulation of gene transcription. One family of guanine nucleotide exchange factors for Rho proteins (RhoGEFs) provides a direct pathway for regulation of RhoA by cell surface receptors coupled to heterotrimeric G proteins. Some of these RhoGEFs also contain RGS domains that can attenuate signaling by the G12 and G13 proteins. The regulation provided by these RhoGEFs is defined by their selective regulation by specific G proteins, phosphorylation by kinases, and potential localization with signaling partners. Evidence of their physiological importance is derived from gene knockouts in Drosophila and mice. Current understanding of the basic regulatory mechanisms of these RhoGEFs is discussed. An overview of identified interactions with other signaling proteins suggests the growing spectrum of their involvement in numerous signaling pathways.
Abstract: 2006 Publication year: 2006 Source:Advances in Protein Chemistry, Volume 73
The β‐form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the β‐conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X‐ray crystallography studies have revealed the high incidence of the β‐fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the β‐fold, summarize the many different new forms of β‐structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.
Abstract: 2006 Publication year: 2006 Source:Advances in Protein Chemistry, Volume 73
It appears that fiber‐forming proteins are not an exclusive group but that, with appropriate conditions, many proteins can potentially aggregate and form fibrils; though only certain proteins, for example, amyloids and silks, do so under normal physiological conditions. Even so, this suggests a ubiquitous aggregation mechanism in which the protein environment is at least as important as the sequence. An ideal model system in which forced and natural aggregation has been observed is silk. Silks have evolved specifically to readily form insoluble ordered structures with a wide range of structural functionality. The animal, be it silkworm or spider, will produce, store, and transport high molecular weight proteins in a complex environment to eventually allow formation of silk fibers with a variety of mechanical properties. Here we review fiber formation and its prerequisites, and discuss the mechanism by which the animal facilitates and modulates silk assembly to achieve controlled protein aggregation.
Abstract: 2006 Publication year: 2006 Source:Advances in Protein Chemistry, Volume 73
β‐Rolls and β‐helices belong to a larger group of topologically similar proteins with solenoid folds: because their regular secondary structure elements are exclusively β‐strands, they are referred to as β‐solenoids. The number of β‐solenoids whose structures are known is now large enough to support a systematic analysis. Here we survey the distinguishing structural features of β‐solenoids, also documenting their notable diversity. Appraisal of these structures suggests a classification based on handedness, twist, oligomerization state, and coil shape. In addition, β‐solenoids are distinguished by the number of chains that wind around a common axis: the majority are single‐stranded but there is a recently discovered subset of triple‐stranded β‐solenoids. This survey has revealed some relationships of the amino acid sequences of β‐solenoids with their structures and functions—in particular, the repetitive character of the coil sequences and conformations that recur in tracts of tandem repeats. We have proposed the term β‐arc for the distinctive turns found in β‐solenoids and β‐arch for the corresponding strand‐turn‐strand motifs. The evolutionary mechanisms underlying these proteins are also discussed. This analysis has direct implications for sequence‐based detection, structural prediction, and de novo design of other β‐solenoid proteins. The abundance of virulence factors, toxins and allergens among β‐solenoids, as well as commonalities of β‐solenoids with amyloid fibrils, imply that this class of folds may have a broader role in human diseases than was previously recognized. Thus, identification of genes with putative β‐solenoid domains promises to be a fertile direction in the search for viable targets in the development of new antibiotics and vaccines.
Abstract: 2006 Publication year: 2006 Source:Advances in Protein Chemistry, Volume 73
A distinctive family of β‐structured folds has recently been described for fibrous proteins from viruses. Virus fibers are usually involved in specific host‐cell recognition. They are asymmetric homotrimeric proteins consisting of an N‐terminal virus‐binding tail, a central shaft or stalk domain, and a C‐terminal globular receptor‐binding domain. Often they are entirely or nearly entirely composed of β‐structure. Apart from their biological relevance and possible gene therapy applications, their shape, stability, and rigidity suggest they may be useful as blueprints for biomechanical design. Folding and unfolding studies suggest their globular C‐terminal domain may fold first, followed by a “zipping‐up” of the shaft domains. The C‐terminal domains appear to be important for registration because peptides corresponding to shaft domains alone aggregate into nonnative fibers and/or amyloid structures. C‐terminal domains can be exchanged between different fibers and the resulting chimeric proteins are useful as a way to solve structures of unknown parts of the shaft domains. The following natural triple β‐stranded fibrous folds have been discovered by X‐ray crystallography: the triple β‐spiral, triple β‐helix, and T4 short tail fiber fold. All have a central longitudinal hydrophobic core and extensive intermonomer polar and nonpolar interactions. Now that a reasonable body of structural and folding knowledge has been assembled about these fibrous proteins, the next challenge and opportunity is to start using this information in medical and industrial applications such as gene therapy and nanotechnology.
Abstract: 2006 Publication year: 2006 Source:Advances in Protein Chemistry, Volume 73
Infectious proteins (prions) became an important medical issue when they were identified as agents of the transmissible spongiform encephalopathies. More recently, prions have been found in fungi and their investigation has been facilitated by greater experimental tractability. In each case, the normal form of the prion protein may be converted into the infectious form (the prion itself) in an autocatalytic process; conversion may either occur spontaneously or by transmission from an already infected cell. Four fungal prion proteins have been studied in some depth—Ure2p, Sup35p, and Rnq1p of Saccharomyces cerevisiae and HET‐s of Podospora anserina. Each has a “prion domain” that governs infectivity and a “functional domain” that contributes the protein's activity in a wild‐type cell, if it has one. This activity is repressed in prion‐infected cells for loss‐of‐activity prions, [URE3] (the prion of Ure2p) and [PSI] (the prion of Sup35p). For gain‐of‐activity prions, [PIN] (the prion of Rnq1p) and [Het‐s] (the prion of HET‐s), the prion domain is also involved in generating a new activity in infected cells. In prion conversion, prion domains polymerize into an amyloid filament, switching from a “natively unfolded” conformation into an amyloid conformation (stable, protease‐resistant, rich in cross‐β structure). For Ure2p and probably also Sup35p, the functional domain retains its globular fold but is inactivated by a steric mechanism. We review the evidence on which this scenario is based with emphasis on filament structure, summarizing current experimental constraints and appraising proposed models. We conclude that the parallel superpleated β‐structure and a specific β‐helical formulation are valid candidates while other proposals are excluded. In both the Ure2p and Sup35p systems, prion domain amyloid filaments exhibit polymorphic variation. However, once a certain structure is nucleated, it is maintained throughout that filament. Electron microscopy of several Ure2p‐related constructs indicates that the basis for polymorphism lies mainly if not entirely in the prion domain. Filament polymorphism appears to underlie the phenomenon of prion “variants” which differ in the severity of their phenotype, that is, for Ure2p and Sup35p, the stringency with which their activity is switched off. We discuss a possible structural basis for this phenomenon.
Abstract: 2006 Publication year: 2006 Source:Advances in Protein Chemistry, Volume 73
A conformational change from the α‐helical, cellular form of prion to the β‐sheet, scrapie (infectious) form is the central event for prion replication. The folding mechanism underlying this conformational change has not yet been deciphered. Here, we review prion pathology and summarize X‐ray fiber and powder diffraction studies on the N‐terminal fragments of prion protein and on short sequences that initiate the β‐assembly for various fibrils, including poly(L‐alanine) and poly(L‐glutamine). We discuss how the quarter‐staggered β‐sheet assembly (like in polyalanine) and polar‐zipper β‐sheet formation (like in polyglutamine) may be involved in the formation of the scrapie form of prion.
Abstract: 2006 Publication year: 2006 Source:Advances in Protein Chemistry, Volume 73
Extracellular amyloid deposits are present in a variety of diseases. They contain amyloid fibrils that arise from the association of proteins or peptides. At the molecular level, all these fibrils share a common assembly principle based on a conformational change of the protein precursor leading to the formation of a cross‐β sheet structure. The smallest observed fibrils in vitro, often called protofibrils, are 4–5 nm in diameter. An amyloid fibril is generally composed of several of these protofibrils and may adopt different morphologies such as ribbons, sheets, or multistranded cables. This polymorphism was observed with many different amyloid‐forming peptides and proteins using electron microscopy. The need to understand the molecular origin of this effect as well as the desire to find inhibitors of fibril formation has driven researchers toward the dissection of amyloid fibril assembly pathways. We review the current knowledge on amyloid polymorphism and discuss recent findings in the field concerning amyloid fibril assembly pathways and cytotoxicity mechanisms.
Abstract: 2006 Publication year: 2006 Source:Advances in Protein Chemistry, Volume 73
Amyloid fibrils are elongated, insoluble protein aggregates deposited in vivo in amyloid diseases, and amyloid‐like fibrils are formed in vitro from soluble proteins. Both of these groups of fibrils, despite differences in the sequence and native structure of their component proteins, share common properties, including their core structure. Multiple models have been proposed for the common core structure, but in most cases, atomic‐level structural details have yet to be determined. Here we review several structural models proposed for amyloid and amyloid‐like fibrils and relate features of these models to the common fibril properties. We divide models into three classes: Refolding, Gain‐of‐Interaction, and Natively Disordered. The Refolding models propose structurally distinct native and fibrillar states and suggest that backbone interactions drive fibril formation. In contrast, the Gain‐of‐Interaction models propose a largely native‐like structure for the protein in the fibril and highlight the importance of specific sequences in fibril formation. The Natively Disordered models have aspects in common with both Refolding and Gain‐of‐Interaction models. While each class of model suggests explanations for some of the common fibril properties, and some models, such as Gain‐of‐Interaction models with a cross‐β spine, fit a wider range of properties than others, no one class provides a complete explanation for all amyloid fibril behavior.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
Hydrogen bonds are an important contributor to free energies of biological macromolecules and macromolecular complexes, and hence an accurate description of these interactions is important for progress in biomolecular modeling. A simple description of the hydrogen bond is based on an electrostatic dipole–dipole interaction involving hydrogen‐donor and acceptor–acceptor base dipoles, but the physical nature of hydrogen bond formation is more complex. At the most fundamental level, hydrogen bonding is a quantum mechanical phenomenon with contributions from covalent effects, polarization, and charge transfer. Recent experiments and theoretical calculations suggest that both electrostatic and covalent components determine the properties of hydrogen bonds. Likely, the level of rigor required to describe hydrogen bonding will depend on the problem posed. Current approaches to modeling hydrogen bonds include knowledge‐based descriptions based on surveys of hydrogen bond geometries in structural databases of proteins and small molecules, empirical molecular mechanics models, and quantum mechanics‐based electronic structure calculations. Ab initio calculations of hydrogen bonding energies and geometries accurately reproduce energy landscapes obtained from the distributions of hydrogen bond geometries observed in protein structures. Orientation‐dependent hydrogen bonding potentials were found to improve the quality of protein structure prediction and refinement, protein–protein docking, and protein design.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
The contribution of backbone‐backbone hydrogen bonds (H‐bonds) to protein folding energetics has been controversial. This is due, at least in part, to the inability to perturb backbone‐backbone H‐bonds by traditional methods of protein mutagenesis. Recently, however, protein backbone mutagenesis has become possible with the development of chemical and biological methods to replace individual amides in the protein backbone with esters. Here, we review the use of amide‐to‐ester mutation as a tool to evaluate the contribution of backbone‐backbone H‐bonds to protein folding kinetics and thermodynamics.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
This chapter discusses methods for modeling electronic polarization in proteins and protein–ligand complexes. Two different approaches are considered: explicit incorporation of polarization into a molecular mechanics force field and the use of mixed quantum mechanics/molecular mechanics methods to model polarization in a restricted region of the protein or protein–ligand complex. A brief description is provided of the computational methodology and parameterization protocols and then results from two preliminary studies are presented. The first study employs quantum mechanics/molecular mechanics (QM/MM) methods to improve the accuracy of protein–ligand docking; here, incorporation of polarization is shown to dramatically improve the robustness of the accuracy of structural prediction of the protein‐ligand docking by enabling qualitative improvement in the selection of the correct hydrogen bonding patterns of the docked ligand. The second study discusses a 2‐ns simulation of bovine pancreatic trypsin inhibitor (BPTI) in water using a variety of fixed charge and polarizable models for both the protein and the solvent, analyzing observed root mean square deviations (RMSD), intraprotein hydrogen bonding, and water structure and dynamics. All of these efforts are in a relatively early stage of development, the results are encouraging in that stable methods have been developed, and significant effects of polarization are seen and (in the case of the QM/MM‐based docking) improvements have been validated as compared to experiment. With regard to accuracy and robustness of full simulations, a great deal more work needs to be done to quantitate and improve the present models.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
This chapter reviews formulation and parametrization of molecular mechanics force fields with special attention to technical and inherent problems. Most striking among the shortcomings is the inadequacy of the simple point charge description as a means to describe energy and forces of interactions between polar molecules and between polar groups in macromolecules, including hydrogen bonds. The current state of efforts to improve the description of polar interactions is discussed.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
Hydrogen bonding underlies the structure of water and all biochemical processes in aqueous medium. Analysis of modern ab initio wave functions in terms of natural bond orbitals (NBOs) strongly suggests the resonance‐type “charge transfer” (CT) character of H‐bonding, contrary to the widely held classical‐electrostatic viewpoint that underlies current molecular dynamics (MD) modeling technology. Quantum cluster equilibrium (QCE) theory provides an alternative ab initio‐based picture of liquid water that predicts proton‐ordered two‐coordinate H‐bonding patterns, dramatically different from the ice‐like picture of electrostatics‐based MD simulations. Recent X‐ray absorption and Raman scattering experiments of Nilsson and co‐workers confirm the microstructural two‐coordinate picture of liquid water. We show how such cooperative “unsaturated” ring/chain topologies arise naturally from the fundamental resonance–CT nature of B:⋯HA hydrogen bonding, which is expressed in NBO language as n B → σ AH * intermolecular delocalization from a filled lone pair n B of the Lewis base (B:) into the proximal antibond σ AH * of the Lewis acid (HA). Stabilizing n O → σ OH * orbital delocalization, equivalent to partial mixing of resonance structures H2O:⋯HOH ⟷ H3O+ ⋯−:OH, is thereby seen to be the electronic origin of general enthalpic and entropic propensities that favor relatively small cyclic clusters such as water pentamers W5c in the QCE liquid phase. We also discuss the thermodynamically competitive three‐coordinate clusters (e.g., icosahedral water buckyballs, W24), which appear to play a role in hydrophobic solvation phenomena. We conclude with suggestions for incorporating resonance–CT aspects of H‐bonding into empirical MD simulation potentials in a computationally tractable manner.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
The energetic cost of partitioning peptide bonds into membrane bilayers is prohibitive unless the peptide bonds participate in hydrogen bonds. However, even then there is a significant free energy penalty for dehydrating the peptide bonds that can only be overcome by favorable hydrophobic interactions. Membrane protein structure formation is thus dominated by hydrogen bonding interactions, which is the subject of this review.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
Since biomolecules exist in aqueous and membrane environments, the accurate modeling of solvation, and hydrogen bonding interactions in particular, is essential for the exploration of structure and function in theoretical and computational studies. In this chapter, we focus on alternatives to explicit solvent models and discuss recent advances in generalized Born (GB) implicit solvent theories. We present a brief review of the successes and shortcomings of the application of these theories to biomolecular problems that are strongly linked to backbone H-bonding and electrostatics. This discussion naturally leads us to explore existing areas for improvement in current GB theories and our approach towards addressing a number of the key issues that remain in the refinement of these models. Specifically, the critical importance of balancing solvation forces and intramolecular forces in GB models is illustrated by examining the influence of backbone hydrogen bond strength and backbone dihedral energetics on conformational equilibria of small peptids.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
The α‐helix was the first proposed and experimentally confirmed secondary structure. The elegant simplicity of the α‐helical structure, stabilized by hydrogen bonding between the backbone carbonyl oxygen and the peptide amide four residues away, has captivated the scientific community. In proteins, α‐helices are also stabilized by the so‐called capping interactions that occur at both the C‐ and the N‐termini of the helix. This chapter provides a brief historical overview of the thermodynamic studies of the energetics of helix formation, and reviews recent progress in our understanding of the thermodynamics of helix formation.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 72
Proteins and peptides in solution or in vivo share properties with both liquids and solids. More often than not, they are studied using the liquid paradigm rather than that of a solid. Studies of molecular crystals illustrate how the use of a solid paradigm may change the way that we consider these important molecules. Cooperative interactions, particularly those involving H‐bonding, play much more important roles in the solid than in the liquid paradigms, as molecular crystals clearly illustrate. Using the solid rather than the liquid paradigm for proteins and peptides includes these cooperative interactions while application of the liquid paradigm tends to ignore or minimize them. Use of the solid paradigm has important implications for basic principles that are often implied about peptide and protein chemistry, such as the importance of entropy in protein folding and the nature of the hydrophobic effect. Understanding the folded states of peptides and proteins (especially α‐helices) often requires the solid paradigm, whereas understanding unfolded states does not. Both theoretical and experimental studies of the energetics of protein and peptide folding require comparison to a suitable standard. Our perspective on these energetics depends on the reasonable choice of reference. The use of multiple reference states, particularly that of component amino acids in the gas phase, is proposed.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
Cell maintenance, cell division, and whole cell movement of the cell body involve a variety of molecular motors. Many of the myosin and kinesin motors are double-headed. Some of these motors, like myosin in muscle, are nonprocessive, whereas others, such as some double-headed nonmuscle myosins and the kinesins, are processive. Because many of these non-muscle motors may work in isolation, the double-headed motor systems can stay attached to their track by at least one of their two heads at all times so that they and their cargo can progress along the track. Whether the motors are processive or not depends on the careful control of the ATPase cycle. Some motile systems involve the aggregation of globular subunits into filaments. Actin and tubulin polymerization are well-documented examples of this. Finally, some of the proteins that help to process the necessary activities of nucleic acids in cells, the polymerases, also show motile features and are powered by ATP. The chapter summarizes the general structures of (1) myosin IIs, (2) kinesins, and (3) cytoplasmic dyneins—found in association with dynactin; and it discusses the way ATPases produces movement.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
This chapter discusses the major muscle components, the actin and myosin filaments; and it also presents a third set of filaments, the titin filaments, which have remarkable properties and play a central role in integrating the sarcomere structure. The chapter further describes the way these filaments are organized in the muscle repeating unit, the sarcomere, through the cross-linking M-band and Z-band structures. The muscle sarcomere contains the principal contractile proteins, myosin and actin, which on their own can produce force and movement, together with a number of cytoskeletal and regulatory proteins. In a cross-section through the A-bands of vertebrate striated muscles, the myosin filaments lie on a hexagonal lattice, and the actin filaments are at the trigonal points, midway between three mutually adjacent myosin filaments. Vertebrate striated muscle sarcomeres contain two remarkable molecular rulers. One of these is titin, which runs from the Z-band to the M-band and provides the sarcomere with mechanical continuity. The other is nebulin, which is an I-band protein anchored in the Z-band.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
This chapter describes titin, the third myofilament of the sarcomere, and outlines the early history that led to the discovery of titin. The chapter also discusses the functional genomics of titin, including an extensive discussion of differential splicing in the I-band region of the molecule. This region is elastic and the molecular mechanism of this elasticity and the way it can be modulated by differential splicing and posttranslational modifications is reviewed in the chapter. Ultrastructural studies with antibodies specific to titin's I-band region have demonstrated that titin's central I-band region behaves extensibly on myofibrillar stretch. The analysis of titin's primary structure revealed specific motif families within its I-band region that act as molecular springs: (1) tandem Ig segments, (2) the PEVK segment, and (3) the N2B-Us segment. The first two elements are found in both skeletal muscle and cardiac muscle titins, but the N2B-Us is cardiac specific. The chapter discusses the titin-binding proteins, including the possible roles of titin-based protein complexes in cell signaling.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
This chapter provides a brief description of tropomyosin's periodic and aperiodic structural features related to their function. The chapter describes the structure of troponin and provides information on F-actin. The thin filaments of vertebrate striated muscle are periodic structures composed of three proteins with different designs that function together for the regulation of contraction. Tropomyosin displays two types of periodicity. In addition to a long-range ∼40-residue repeat, tropomyosin is the paradigm of the α-helical coiled-coil class of proteins. The seven distinct amino acid positions and associated interactions that are produced from the α-helical coiled-coil provide the basic structural unit of tropomyosin. These elements superimpose on the longer, roughly 40-residue, functional unit of tropomyosin and patterns of residues found both in the core of the coiled-coil and on its surface are repeated seven times in a full-length tropomyosin molecule; and they play a role in the periodic binding of tropomyosin to actin.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
This chapter describes the analysis of the polymorphism of the myosin crossbridge and relates it to the Lymn–Taylor crossbridge cycle. Myosin from muscle (myosin II) consists of two long polypeptide chains (heavy chains) combined with four light chains. In cross-striated muscle, the tails of the molecules pack together to form the thick filaments, while the crossbridges that are ATPases point away from the thick filaments and cyclically interact with the actin filaments, moving them along by a kind of rowing action. The fuel for this process is provided by the hydrolysis of adenosine triphosphate (ATP). There are three primary conformations of the myosin crossbridge that can be associated with states in the Lymn–Taylor cycle. These are—namely, the post-rigor structure, the prepower stroke structure, and the rigor-like state. A comparison of these structures leads to the identification of various important conformationally flexible elements, such as (1) the positions of the converter domain, (2) the kink in the relay helix, and (3) the degree of twist of the central β-sheet. The chapter describes these states and then presents the biochemical and kinetic arguments for assigning them to the positions shown in the Lymn–Taylor cycle.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
This chapter discusses the muscle structure and the time‐resolved events in contracting muscles. The chapter explores various issues involved in the X‐ray diffraction studies of muscles. It describes the structure of vertebrate striated muscles. The vertebrate sarcomere consists of bipolar myosin filaments with threefold rotational symmetry, and myosin heads organized in the bridge regions on three co‐axial roughly helical strands with nine head pairs per turn of the helix, giving a true repeat of 429 A° and an axial separation between crowns of heads averaging ∼143 A°. Relating to the X‐ray diffraction from this assembly, a number of the sarcomere components contribute to its observed patterns. In the A‐band, these include the myosin filament backbone, where the coiled‐coil α‐helical myosin rods pack together; the myosin head arrays in the bridge regions of the myosin filaments; the non‐myosin A‐band proteins titin and C‐protein (MyBP‐C); and the A‐band parts of the actin filaments. Of the basic contributors to the muscle diffraction pattern, two parts of the sarcomere that make a significant contribution at high diffraction angles are (1) the internal structure of the actin monomers in the actin filaments and (2) the backbone of the myosin filaments, where the myosin rods are packed. The chapter also discusses the X‐Ray interference measurements and their implications.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
Microtubules are very dynamic polymers whose assembly and disassembly is determined by whether their heterodimeric tubulin subunits are in a straight or curved conformation. Curvature is introduced by bending at the interfaces between monomers. Assembly and disassembly are primarily controlled by the hydrolysis of guanosine triphosphate (GTP) in a site that is completed by the association of two heterodimers. However, a multitude of associated proteins are able to fine‐tune these dynamics so that microtubules are assembled and disassembled where and when they are required by the cell. We review the recent progress that has been made in obtaining a glimpse of the structural interactions involved.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
Microtubules are the intracellular tracks for two classes of motor proteins: kinesins and dyneins. During the past few years, the motor domain structures of several kinesins from different organisms have been determined by X‐ray crystallography. Compared with kinesins, dyneins are much larger proteins and attempts to crystallize them have failed so far. Structural information about these proteins comes mostly from electron microscopy. In this chapter, we mainly focus on the crystal structures of kinesin motor domains.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
The F‐, V‐, and A‐adenosine triphosphatases (ATPases) represent a family of evolutionarily related ion pumps found in every living cell. They either function to synthesize adenosine triphosphate (ATP) at the expense of an ion gradient or they act as primary ion pumps establishing transmembrane ion motive force at the expense of ATP hydrolysis. The A‐, F‐, and V‐ATPases are rotary motor enzymes. Synthesis or hydrolysis of ATP taking place in the three catalytic sites of the membrane extrinsic domain is coupled to ion translocation across the single ion channel in the membrane‐bound domain via rotation of a central part of the complex with respect to a static portion of the enzyme. This chapter reviews recent progress in the structure determination of several members of the family of F‐, A‐, and V‐ATPases and our current understanding of the rotary mechanism of energy coupling.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
Nematode sperm provide a simple and specialized system for studying the molecular mechanism of amoeboid cell motility. Locomotion is generated by the assembly dynamics of their cytoskeleton, which is based on the major sperm protein (MSP). Protrusive force is generated at the leading edge of the lamellipod by MSP filament formation and bundling, whereas the contractile force that drags the rearward cell body forward is generated by cytoskeleton disassembly. The dynamics of the system can be reconstituted in vitro using cell‐free extracts of Ascaris sperm, in which vesicles derived from the leading edge of the cell can be either pushed or pulled. The addition of ATP to the cell‐free extract initiates MSP filament polymerization and bundling immediately behind the vesicle, and the expansion of the resulting gel pushes the vesicle at rates comparable to those seen in living cells. In contrast, the addition of Yersinia tyrosine phosphatase generates depolymerization and gel contraction that pulls the vesicles. Overall, nematode sperm motility illustrates that cell locomotion can be generated by cytoskeletal dynamics alone without the use of myosin‐like motor proteins.
Abstract: 2005 Publication year: 2005 Source:Advances in Protein Chemistry, Volume 71
DNA polymerases are molecular motors directing the synthesis of DNA from nucleotides. All polymerases have a common architectural framework consisting of three canonical subdomains termed the fingers, palm, and thumb subdomains. Kinetically, they cycle through various states corresponding to conformational transitions, which may or may not generate force. In this review, we present and discuss the kinetic, structural, and single‐molecule works that have contributed to our understanding of DNA polymerase function.
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