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Query: UMLS:C0038454 (stroke)
 147,016 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at approximately 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.
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PMID:Force-dependent stepping kinetics of myosin-V. 1576 64

The power stroke pulling myosin along actin filaments during muscle contraction is achieved by a large rotation ( approximately 60 degrees ) of the myosin lever arm after ATP hydrolysis. Upon binding the next ATP, myosin dissociates from actin, but its ATPase site is still partially open and catalytically off. Myosin must then close and activate its ATPase site while returning the lever arm for the next power stroke. A mechanism for this coupling between the ATPase site and the distant lever arm is determined here by generating a continuous series of optimized intermediates between the crystallographic end-states of the recovery stroke. This yields a detailed structural model for communication between the catalytic and the force-generating regions that is consistent with experimental observations. The coupling is achieved by an amplifying cascade of conformational changes along the relay helix lying between the ATPase and the domain carrying the lever arm.
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PMID:Structural mechanism of the recovery stroke in the myosin molecular motor. 1586 18

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single alpha-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the full-length predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.
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PMID:The predicted coiled-coil domain of myosin 10 forms a novel elongated domain that lengthens the head. 1603 12

Myosin generates force by a rotation of its lever arm. Crystal structures of myosin II indicate an unloaded working stroke of 10-12 nm, a range confirmed by recent x-ray interference experiments. However, when an actin filament, held between two weakly, optically trapped beads is made to interact with a single head of skeletal myosin, the bead displacements have often been reported as having a mean value of 5-6 nm, a value that is commonly interpreted as the working stroke. In general, the observed displacement is not expected to be equal to the working stroke because the kinetics of the stroke is necessarily strain-dependent: this effect biases the frequency of binding events to different actin sites so that displacements smaller than the working stroke are preferentially selected. Our analysis is tailored to current trap experiments, in which the time resolution is insufficient to detect pre-rigor states. If the preceding transitions are in equilibrium, the mean displacement is zero, contrary to observations in the presence of ATP. However, under ATP-cycling conditions, we find that the mean displacement is deflated to 0.3-0.7 of the true working stroke, depending on the equilibrium constant of the stroke and the rate at which the first myosin product state can detach from actin. The primary working stroke of processive myosin motors as measured by optical trapping is similarly uncertain.
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PMID:Reconciling the working strokes of a single head of skeletal muscle myosin estimated from laser-trap experiments and crystal structures. 1642 90

We have used electron paramagnetic probes attached to the ribose of ATP (SL-ATP) to monitor conformational changes in the nucleotide pocket of myosin. Spectra for analogs bound to myosin in the absence of actin showed a high degree of immobilization, indicating a closed nucleotide pocket. In the Actin.Myosin.SL-AMPPNP, Actin.Myosin.SL-ADP.BeF(3), and Actin.Myosin.SL-ADP.AlF(4) complexes, which mimic weakly binding states near the beginning of the power stroke, the nucleotide pocket remained closed. The spectra of the strongly bound Actin.Myosin.SL-ADP complex consisted of two components, one similar to the closed pocket and one with increased probe mobility, indicating a more open pocket, The temperature dependence of the spectra showed that the two conformations of the nucleotide pocket were in equilibrium, with the open conformation more favorable at higher temperatures. These results, which show that opening of the pocket occurs only in the strongly bound states, appear reasonable, as this would tend to keep ADP bound until the end of the power stroke. This conclusion also suggests that force is initially generated by a myosin with a closed nucleotide pocket.
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PMID:Dynamics of the nucleotide pocket of myosin measured by spin-labeled nucleotides. 1702 39

Myosin is the molecular motor in muscle-binding actin and executing a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. A green fluorescent protein (GFP)-tagged human cardiac myosin regulatory light chain (HCRLC) was constructed to study in situ lever arm orientation one molecule at a time by polarized fluorescence emitted from the GFP probe. The recombinant protein physically and functionally replaced the native RLC on myosin lever arms in the thick filaments of permeabilized skeletal muscle fibers. Detecting single molecules in fibers where myosin concentration reaches 300 microM is accomplished using total internal reflection fluorescence microscopy. With total internal reflection fluorescence, evanescent field excitation, supercritical angle fluorescence detection, and CCD detector pixel size limits detection volume to just a few attoliters. Data analysis manages both the perturbing effect of the TIR interface on probe emission and the effect of high numerical aperture collection of light. The natural myosin concentration gradient in a muscle fiber allows observation of fluorescence polarization from C-term GFP-tagged HCRLC exchanged myosin from regions in the thick filament containing low and high myosin concentrations. In rigor, cross-bridges at low concentration at the end of the thick filament maintain GFP dipole moments at two distinct polar angles relative to the fiber symmetry axis. The lower angle, where the dipole is nearly parallel to fiber axis, is more highly populated than the alternative, larger angle. Cross-bridges at higher concentration in the center of the thick filament are oriented in a homogeneous band at approximately 45 degrees to the fiber axis. The data suggests molecular crowding impacts myosin conformation, implying mutual interactions between cross-bridges alter how the muscle generates force. The GFP-tagged RLC is a novel probe to assess single-lever-arm orientation characteristics in situ.
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PMID:GFP-tagged regulatory light chain monitors single myosin lever-arm orientation in a muscle fiber. 1751 76

Myosin 2 is the molecular motor in muscle. It binds actin and executes a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. Myosin 2 has evolved to function optimally under crowded conditions where rates and equilibria of macromolecular reactions undergo major shifts relative to those measured in dilute solution. Hence, an important research objective is to detect in situ the lever arm orientation. Single-molecule measurements are preferred because they clarify ambiguities that are unavoidable with ensemble measurements; however, detecting single molecules in the condensed tissue medium where the myosin concentration exceeds 100 muM is challenging. A myosin light chain (MLC) tagged with photoactivatable green fluorescent protein (PAGFP) was constructed. The recombinant MLC physically and functionally replaced native MLC on the myosin lever arm in a permeabilized skeletal muscle fiber. Probe illumination volume was minimized using total internal reflection fluorescence microscopy, and PAGFP was sparsely photoactivated such that polarized fluorescence identified a single probe orientation. Several physiological states of the muscle fiber were characterized, revealing two distinct orientation populations in all states called straight and bent conformations. Conformation occupancy probability varies among fiber states with rigor and isometric contraction at extremes where straight and bent conformations predominate, respectively. Comparison to previous work on single rigor cross-bridges at the A-band periphery where the myosin concentration is low suggests molecular crowding in the A-band promotes occupancy of the straight myosin conformation [Burghardt, T. P., et al. (2007) Biophys. J. 93, 2226]. The latter may have a role in contraction because it provides additional free energy favoring completion of the cross-bridge power stroke.
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PMID:Single myosin lever arm orientation in a muscle fiber detected with photoactivatable GFP. 1912 92

Actin and myosin form the molecular motor in muscle. Myosin is the enzyme performing ATP hydrolysis under the allosteric control of actin such that actin binding initiates product release and force generation in the myosin power stroke. Non-equilibrium Monte Carlo molecular dynamics simulation of the power stroke suggested that a structured surface loop on myosin, the C-loop, is the actin contact sensor initiating actin activation of the myosin ATPase. Previous experimental work demonstrated C-loop binds actin and established the forward and reverse allosteric link between the C-loop and the myosin active site. Here, smooth muscle heavy meromyosin C-loop chimeras were constructed with skeletal (sCl) and cardiac (cCl) myosin C-loops substituted for the native sequence. In both cases, actin-activated ATPase inhibition is indicated mainly by the lower V(max). In vitro motility was also inhibited in the chimeras. Motility data were collected as a function of myosin surface density, with unregulated actin, and with skeletal and cardiac isoforms of tropomyosin-bound actin for the wild type, cCl, and sCl. Slow and fast subpopulations of myosin velocities in the wild-type species were discovered and represent geometrically unfavorable and favorable actomyosin interactions, respectively. Unfavorable interactions are detected at all surface densities tested. Favorable interactions are more probable at higher myosin surface densities. Cardiac tropomyosin-bound actin promotes the favorable actomyosin interactions by lowering the inhibiting geometrical constraint barriers with a structural effect on actin. Neither higher surface density nor cardiac tropomyosin-bound actin can accelerate motility velocity in cCl or sCl, suggesting the element initiating maximal myosin activation by actin resides in the C-loop.
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PMID:The myosin C-loop is an allosteric actin contact sensor in actomyosin. 1940 46

Myosin II is a molecular motor that converts chemical to mechanical energy and enables muscle operations. After a power stroke, a recovery transition completes the cycle and returns the molecular motor to its prestroke state. Atomically detailed simulations in the framework of the Milestoning theory are used to calculate kinetics and mechanisms of the recovery stroke. Milestoning divides the process into transitions between hyper-surfaces (Milestones) along a reaction coordinate. Decorrelation of dynamics between sequential Milestones is assumed, which speeds up the atomically detailed simulations by a factor of millions. Two hundred trajectories of myosin with explicit water solvation are used to sample transitions between sequential pairs of Milestones. Collective motions of hundreds of atoms are described at atomic resolution and at the millisecond time scale. The experimentally measured transition time of about a millisecond is in good agreement with the computed time. The simulations support a sequential mechanism. In the first step the P-loop and switch 2 close on the ATP and in the second step the mechanical relaxation is induced via the relay and the SH1 helices. We propose that the entropy of switch 2 helps to drive the power stroke. Secondary structure elements are progressing through a small number of discrete states in a network of activated transitions and are assisted by side chain flips between rotameric states. The few-state sequential mechanism is likely to enhance the efficiency of the relaxation reducing the probability of off-pathway intermediates.
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PMID:Atomically detailed simulation of the recovery stroke in myosin by Milestoning. 2019 70

Using optical trapping and fluorescence imaging techniques, we measured the step size and stiffness of single skeletal myosins interacting with actin filaments and arranged on myosin-rod cofilaments that approximate myosin mechanics during muscle contraction. Stiffness is dramatically lower for negatively compared to positively strained myosins, consistent with buckling of myosin's subfragment 2 rod domain. Low stiffness minimizes drag of negatively strained myosins during contraction at loaded conditions. Myosin's elastic portion is stretched during active force generation, reducing apparent step size with increasing load, even though the working stroke is approximately constant at about 8 nanometers. Taking account of the nonlinear nature of myosin elasticity is essential to relate myosin's internal structural changes to physiological force generation and filament sliding.
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PMID:Nonlinear elasticity and an 8-nm working stroke of single myosin molecules in myofilaments. 2068 17


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