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David D Thomas - One of the best experts on this subject based on the ideXlab platform.
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FRET and optical trapping reveal mechanisms of actin activation of the Power Stroke and phosphate release in myosin V
The Journal of biological chemistry, 2020Co-Authors: Laura K. Gunther, David D Thomas, Wanjian Tang, John A. Rohde, Joseph A. Cirilo, Christopher P. Marang, Brent Scott, Edward P. Debold, Christopher M. YengoAbstract:Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery Stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (Power Stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–Power Stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working Stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the Power Stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a Power Stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.
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direct measurements of the coordination of lever arm swing and the catalytic cycle in myosin v
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Darshan V. Trivedi, Jonathan P. Davis, Anja M. Swenson, Joseph M Muretta, David D Thomas, Christopher M. YengoAbstract:Myosins use a conserved structural mechanism to convert the energy from ATP hydrolysis into a large swing of the force-generating lever arm. The precise timing of the lever arm movement with respect to the steps in the actomyosin ATPase cycle has not been determined. We have developed a FRET system in myosin V that uses three donor–acceptor pairs to examine the kinetics of lever arm swing during the recovery and Power Stroke phases of the ATPase cycle. During the recovery Stroke the lever arm swing is tightly coupled to priming the active site for ATP hydrolysis. The lever arm swing during the Power Stroke occurs in two steps, a fast step that occurs before phosphate release and a slow step that occurs before ADP release. Time-resolved FRET demonstrates a 20-A change in distance between the pre- and postPower Stroke states and shows that the lever arm is more dynamic in the postPower Stroke state. Our results suggest myosin binding to actin in the ADP.Pi complex triggers a rapid Power Stroke that gates the release of phosphate, whereas a second slower Power Stroke may be important for mediating strain sensitivity.
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Direct real-time detection of the structural and biochemical events in the myosin Power Stroke.
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Joseph M Muretta, John A. Rohde, Daniel O. Johnsrud, Sinziana Cornea, David D ThomasAbstract:A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)2FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin’s Power Stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the Power Stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which Power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.
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amplitude of the actomyosin Power Stroke depends strongly on the isoform of the myosin essential light chain
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Piyali Guhathakurta, Ewa Prochniewicz, David D ThomasAbstract:We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating Power Stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (Power Stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (Power Stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger Power Stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.
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Direct real-time detection of the actin-activated Power Stroke within the myosin catalytic domain
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Joseph M Muretta, Karl J. Petersen, David D ThomasAbstract:We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated Power Stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prePower Stroke state of the relay helix and a straight postPower Stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postPower Stroke state, preventing reversal of the Power Stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.
Hideo Higuchi - One of the best experts on this subject based on the ideXlab platform.
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Analysis of spontaneous oscillations for a three-state Power-Stroke model.
Physical Review E, 2017Co-Authors: Takumi Washio, Toshiaki Hisada, Seine A. Shintani, Hideo HiguchiAbstract:Our study considers the mechanism of the spontaneous oscillations of molecular motors that are driven by the Power Stroke principle by applying linear stability analysis around the stationary solution. By representing the coupling equation of microscopic molecular motor dynamics and mesoscopic sarcomeric dynamics by a rank-1 updated matrix system, we derived the analytical representations of the eigenmodes of the Jacobian matrix that cause the oscillation. Based on these analytical representations, we successfully derived the essential conditions for the oscillation in terms of the rate constants of the Power Stroke and the reversal Stroke transitions of the molecular motor. Unlike the two-state model, in which the dependence of the detachment rates on the motor coordinates or the applied forces on the motors plays a key role for the oscillation, our three-state Power Stroke model demonstrates that the dependence of the rate constants of the Power and reversal Strokes on the strains in the elastic elements in the motor molecules plays a key role, where these rate constants are rationally determined from the free energy available for the Power Stroke, the stiffness of the elastic element in the molecular motor, and the working Stroke size. By applying the experimentally confirmed values to the free energy, the stiffness, and the working Stroke size, our numerical model reproduces well the experimentally observed oscillatory behavior. Furthermore, our analysis shows that two eigenmodes with real positive eigenvalues characterize the oscillatory behavior, where the eigenmode with the larger eigenvalue indicates the transient of the system of the quick sarcomeric lengthening induced by the collective reversal Strokes, and the smaller eigenvalue correlates with the speed of sarcomeric shortening, which is much slower than lengthening. Applying the perturbation analyses with primal physical parameters, we find that these two real eigenvalues occur on two branches derived from a merge point of a pair of complex-conjugate eigenvalues generated by Hopf bifurcation.
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The Power Stroke Distance of Human Cytoplasmic Dynein
Biophysical Journal, 2017Co-Authors: Yoshimi Kinoshita, Motoshi Kaya, Taketoshi Kambara, Kaori Nishikawa, Hideo HiguchiAbstract:Cytoplasmic dynein is a motor protein moving along microtubules, and plays important roles in vesicle transport and mitosis. To understand the molecular mechanism of dynein motility, we measured the efficiency of FRET from dynein ring-BFP to linker-GFP, and the displacement driven by single-headed dynein interacting with microtubules by optical tweezers. The efficiency and the apparent Stroke displacement depend on ATP concentration. The low efficiency and displacement at low ATP concentration indicate no conformational change of dynein at no ATP binding (apo) state that is predominant at low ATP concentration. With increasing ATP concentration, population of apo state decreases and that of the pre-Power Stroke state such as dynein ADP-Pi should increase. Dynein at the pre-Power Stroke state will generate the Power Stroke at binding to microtubule. Therefore, the displacement increased with ATP concentration. This is the first obtained interesting result and has not been reported for myosin and kinesin Power Strokes. High FRET efficiency and distance (8-9 nm) at saturated concentration of ATP indicate that the 8-9 nm Power Stroke distance is generated by swing of linker. This is supported by the result that the dynein deleted at the loops interacted with the linker did not generate the Power Stroke. The Power Stroke driven by the conformational change of dynein linker will be the fundamental mechanism of dynein motility.
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mechanism of cooperative force generations between skeletal myosins
Biophysical Journal, 2016Co-Authors: Motoshi Kaya, Yoshiaki Tani, Takumi Washio, Toshiaki Hisada, Hideo HiguchiAbstract:To understand the molecular mechanism of cooperative force generation between skeletal myosin molecules, we measured forces generated by myosin-rod cofilaments, in which approximately 17 myosin molecules interact with single actins at the mixing ratio used in this study. Combined with results from the computational model, three factors are important for synchronization of Power Strokes between myosin motors. First, strain-dependent kinetics are necessary to couple mechanochemical cycles between myosins. Second, multiple Power Stroke states further enhance a chance of Power Stroke synchronization. Finally, the physiological ATP concentration is another important factor to enhance a chance of Power Stroke synchronization, since the strain-dependent transitions accompanied by the first or second Power Stroke are the rate limiting steps at higher ATP concentrations. Consequently, our computational model predicts that most of steps were generated by synchronous executions of Power Strokes between several myosin motors at 1 mM ATP, while they are generated primarily by single myosins. Thus, ensemble average curves of steps obtained from our model were distinctively different between 1 mM and 10 μM ATP. These results were consistent with experimental results, supporting our conclusions.
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intermolecular cooperativity of skeletal myosins enhances force output in myofilaments
Biophysical Journal, 2015Co-Authors: Motoshi Kaya, Yoshiaki Tani, Takumi Washio, Toshiaki Hisada, Hideo HiguchiAbstract:Muscle contraction is Powered by the cyclic interaction of skeletal myosin molecules with actin filaments. Recent experiments suggest cooperative actions between myosin molecules, when part of an ensemble. In order to gain more insight into the mechanism of intermolecular cooperativity between myosins in a myofilament, displacements of actin generated by ∼17 interacting myosins embedded in myosin-rod cofilament were measured by optical tweezers. Results showed stepwise displacements of actin (3-6 nm) under high loads of ∼30 pN at every 1-2 ms. Dwell times gradually increased with increasing loads, which is distinctively different from highly load-dependent characteristics of dwell time observed in single myosins, implying that the number of force generating myosins increases with increasing loads. These results suggest that stepwise displacements may be generated by synchronous force generations of myosin molecules. In order to elucidate potential mechanisms of synchronous actions between myosins, we developed the simulation model, consisting of 17 myosins arranged in series with six transition states defined during actomyosin mechanochemical cycles. We have two types of model, implemented with either one or two Power Strokes. Interestingly, two Power Stroke model generates stepwise movements of actin at loads up to 30 pN, but one Power Stroke model generates forces up to 20 pN. These differences in force output are attributed to modulation of number of synchronous Power Stroke motors. The numbers of synchronous Power Stroke motors increase with increasing loads from 1.5 to 3 molecules in two Power Stroke model on average, while they are virtually the same in one Power Stroke model. Combined with results from both in-vitro and in-silico experiments, multiple Power Stroke states associated with strain-dependent kinetics are essential for synchronous force generations between myosins, that is a key feature for force enhancement of myosin ensembles.
Dilson E Rassier - One of the best experts on this subject based on the ideXlab platform.
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millisecond conformational dynamics of skeletal myosin ii Power Stroke studied by high speed atomic force microscopy
ACS Nano, 2021Co-Authors: Oleg S Matusovsky, Noriyuki Kodera, Caitlin Maceachen, Toshio Ando, Yushu Cheng, Dilson E RassierAbstract:Myosin-based molecular motors are responsible for a variety of functions in the cells. Myosin II is ultimately responsible for muscle contraction and can be affected by multiple mutations, that may lead to myopathies. Therefore, it is essential to understand the nanomechanical properties of myosin II. Due to the lack of technical capabilities to visualize rapid changes in nonprocessive molecular motors, there are several mechanistic details in the force-generating steps produced by myosin II that are poorly understood. In this study, high-speed atomic force microscopy was used to visualize the actin-myosin complex at high temporal and spatial resolutions, providing further details about the myosin mechanism of force generation. A two-step motion of the double-headed heavy meromyosin (HMM) lever arm, coupled to an 8.4 nm working Stroke was observed in the presence of ATP. HMM heads attached to an actin filament worked independently, exhibiting different lever arm configurations in given time during experiments. A lever arm rotation was associated with several non-stereospecific long-lived and stereospecific short-lived (∼1 ms) HMM conformations. The presence of free Pi increased the short-lived stereospecific binding events in which the Power Stroke occurred, followed by release of Pi after the Power Stroke.
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Pre-Power-Stroke cross-bridges contribute to force transients during imposed shortening in isolated muscle fibers.
PloS one, 2012Co-Authors: Fabio C. Minozzo, Lennart Hilbert, Dilson E RassierAbstract:When skeletal muscles are activated and mechanically shortened, the force that is produced by the muscle fibers decreases in two phases, marked by two changes in slope (P1 and P2) that happen at specific lengths (L1 and L2). We tested the hypothesis that these force transients are determined by the amount of myosin cross-bridges attached to actin and by changes in cross-bridge strain due to a changing fraction of cross-bridges in the pre-Power-Stroke state. Three separate experiments were performed, using skinned muscle fibers that were isolated and subsequently (i) activated at different Ca2+ concentrations (pCa2+ 4.5, 5.0, 5.5, 6.0) (n = 13), (ii) activated in the presence of blebbistatin (n = 16), and (iii) activated in the presence of blebbistatin at varying velocities (n = 5). In all experiments, a ramp shortening was imposed (amplitude 10%Lo, velocity 1 Lo•sarcomere length (SL)•s−1), from an initial SL of 2.5 µm (except by the third group, in which velocities ranged from 0.125 to 2.0 Lo•s−1). The values of P1, P2, L1, and L2 did not change with Ca2+ concentrations. Blebbistatin decreased P1, and it did not alter P2, L1, and L2. We developed a mathematical cross-bridge model comprising a load-dependent Power-Stroke transition and a pre-Power-Stroke cross-bridge state. The P1 and P2 critical points as well as the critical lengths L1 and L2 were explained qualitatively by the model, and the effects of blebbistatin inhibition on P1 were also predicted. Furthermore, the results of the model suggest that the mechanism by which blebbistatin inhibits force is by interfering with the closing of the myosin upper binding cleft, biasing cross-bridges into a pre-Power-Stroke state.
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pre Power Stroke cross bridges contribute to force during stretch of skeletal muscle myofibrils
Proceedings of The Royal Society B: Biological Sciences, 2008Co-Authors: Dilson E RassierAbstract:When activated skeletal muscle is stretched, force increases in two phases. This study tested the hypothesis that the increase in stretch force during the first phase is produced by pre-Power Stroke cross bridges. Myofibrils were activated in sarcomere lengths (SLs) between 2.2 and 2.5 μm, and stretched by approximately 5–15 per cent SL. When stretch was performed at 1 μm s −1 SL −1 , the transition between the two phases occurred at a critical stretch (SL c ) of 8.4±0.85 nm half-sarcomere (hs) −1 and the force (critical force; F c ) was 1.62±0.24 times the isometric force ( n =23). At stretches performed at a similar velocity (1 μm s −1 SL −1 ), 2,3-butanedione monoxime (BDM; 1 mM) that biases cross bridges into pre-Power Stroke states decreased the isometric force to 21.45±9.22 per cent, but increased the relative F c to 2.35±0.34 times the isometric force and increased the SL c to 14.6±0.6 nm hs −1 ( n =23), suggesting that pre-Power Stroke cross bridges are largely responsible for stretch forces.
Tom A Rapoport - One of the best experts on this subject based on the ideXlab platform.
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protein translocation by the seca atpase occurs by a Power Stroke mechanism
The EMBO Journal, 2019Co-Authors: Marco A Catipovic, Benedikt W Bauer, Joseph J Loparo, Tom A RapoportAbstract:SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This Power-Stroke mechanism may be used by other ATPases that move polypeptides.
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Protein translocation by the SecA ATPase occurs by a Power‐Stroke mechanism
The EMBO journal, 2019Co-Authors: Marco A Catipovic, Benedikt W Bauer, Joseph J Loparo, Tom A RapoportAbstract:SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This Power-Stroke mechanism may be used by other ATPases that move polypeptides.
Lev Truskinovsky - One of the best experts on this subject based on the ideXlab platform.
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Power-Stroke-Driven Muscle Contraction
The Mathematics of Mechanobiology, 2020Co-Authors: Raman Sheshka, Lev TruskinovskyAbstract:To show that acto-myosin contraction can be propelled directly through a conformational change, we present in these lecture notes a review of a recently developed approach to muscle contraction where myosin Power-Stroke is interpreted as the main active mechanism. By emphasizing the active role of Power Stroke, the proposed model contributes to building a conceptual bridge between processive and nonprocessive motors.
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Power-Stroke-driven actomyosin contractility
Physical review. E Statistical nonlinear and soft matter physics, 2014Co-Authors: R. Sheshka, Lev TruskinovskyAbstract:In ratchet-based models describing actomyosin contraction the activity is usually associated with actin binding potential while the Power-Stroke mechanism, residing inside myosin heads, is viewed as passive. To show that contraction can be propelled directly through a conformational change, we propose an alternative model where the Power Stroke is the only active mechanism. The asymmetry, ensuring directional motion, resides in steric interaction between the externally driven Power-Stroke element and the passive nonpolar actin filament. The proposed model can reproduce all four discrete states of the minimal actomyosin catalytic cycle even though it is formulated in terms of continuous Langevin dynamics. We build a conceptual bridge between processive and nonprocessive molecular motors by demonstrating that not only the former but also the latter can use structural transformation as the main driving force.
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Mechanics of the Power Stroke in Myosin II
Physical Review E : Statistical Nonlinear and Soft Matter Physics, 2010Co-Authors: Lorenzo Marcucci, Lev TruskinovskyAbstract:Power Stroke in skeletal muscles is a result of a conformational change in the globular portion of the molecular motor myosin II. In this paper we show that the fast tension recovery data reflecting the inner working of the Power Stroke mechanism can be quantitatively reproduced by a Langevin dynamics of a simple mechanical system with only two structural states. The proposed model is a generalization of the two state model of Huxley and Simmons. The main idea is to replace the rigid bistable device of Huxley and Simmons with an elastic bistable snap spring. In this setting the attached configuration of a cross bridge is represented not only by the discrete energy minima but also by a continuum of intermediate states where the fluctuation induced dynamics of the system takes place. We show that such soft-spin approach explains the load dependence of the Power Stroke amplitude and removes the well-known contradiction inside the conventional two state model regarding the time scale of the Power Stroke.
