Movement and force produced by a single myosin head

Molloy, Justin E.; Burns, Julie E.; Kendrick‐Jones, John; Tregear, R. T.; White, D. C. S.

doi:10.1038/378209a0

Cited by 592 publications

(528 citation statements)

References 12 publications

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“…Alternatively, increased fragmentation of actin filaments might result from reduced stability of actin filaments in the presence of phosphate, however, only at low MgATP concentrations. This possibility appears unlikely as in trapping experiments in the three-bead arrangement (42) no evidence for reduced stability of actin filaments was found in the presence of 5 mM P i and M MgATP. 4 By the same line of arguments, the essentially unchanged dwell times of Cy3-EDA-ATP in the presence of phosphate and the reduced number of fluorescent events per field of view upon the addition of phosphate at low Cy3-EDA-ATP concentrations are also inconsistent with one of the concepts proposed by Warshaw et al (25).…”

Section: Discussionmentioning

confidence: 79%

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

Amrute-Nayak

Antognozzi

Scholz

et al. 2008

Journal of Biological Chemistry

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In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times Muscle contraction involves the sliding of actin filaments relative to myosin filaments (1, 2), which is driven by cyclic interactions of the myosin head domains with the actin filaments, powered by hydrolysis of ATP. During each ATP-hydrolysis cycle chemical energy of ATP hydrolysis is transformed into mechanical work by a multistep power-stroke which drives the actin filaments several nanometers past the myosin filaments. The power-stroke is associated with the release of the ATP hydrolysis products, P i (inorganic phosphate) and ADP, from the active site (3). It is generally believed that P i release from the AM⅐ADP⅐P i 3 complex is closely related to the initiation of the power-stroke (4) and to the transition of the myosin head domain from states of weak and non-stereospecific actin binding to states of strong and stereospecific binding to actin (5-7). Because of the assumed close relation between power stroke and the release of inorganic phosphate from the myosin head domain, studying the effects of inorganic phosphate on contracting muscle fibers became a main element to elucidate the relation between P i release and force generation. Effects of P i were examined on isometric force (4, 8 -14) and unloaded shortening velocity (9, 13, 15) as well as on force transients in response to release of P i from caged P i (16,17). As a result of these studies it was proposed that inhibition of active force by inorganic phosphate results from rebinding of P i to the AM⅐ADP intermediate that is formed after P i release (cf. Scheme 1). Thus, the release of phosphate is reversed and a strong binding force generating cross-bridges in the AM⅐ADP state are reversed to a weak binding non-force generating AM⅐ADP⅐P i state (the AM⅐ADP⅐Pi I state in Scheme 1) that is in rapid equilibrium with the detached M⅐ADP⅐P i I intermediate. To fully account for the observed steady state kinetic data and the force transients recorded upon release of P i from caged P i , it was proposed that a strong binding AM⅐ADP⅐P i intermediate (the AM⅐AD...

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Section: Discussionmentioning

confidence: 79%

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

Amrute-Nayak

Antognozzi

Scholz

et al. 2008

Journal of Biological Chemistry

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“…Single molecule mechanical measurements have suggested that the cross-bridge stroke is of the order of 4-17 nm [1][2][3], which is compatible with the conventional model for the crossbridge cycle [5]. However, there is no direct evidence of how many such strokes may be derived from the hydrolysis of a single ATP molecule.…”

Section: Introductionmentioning

confidence: 86%

“…A fundamental goal in understanding the mechanism of this process is to determine whether the ATPase activity is tightly coupled to the mechanical cycle of cross-bridge action. To this end, recent eflbrt has been directed towards in vitro motility assays where mechanical events have now been detected at the level of single molecular interactions between actin and myosin [1][2][3]. Likewise, the sensitivity of ATPase assays have been improved so that the long-lived myosin product complex (M.ADEP~) can be detected at the single molecule level [4].…”

Section: Introductionmentioning

confidence: 99%

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

Conibear

Bagshaw

1996

FEBS Letters

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The kinetics of nucleotide release have been measured at the level of isolated synthetic myosin filaments. This was achieved by displacing a rhodamine nucleotide analog from a filament by flash photolysis of caged-ATP with selective observation using total internal reflectance fluorescence microscopy. The procedure gave improved time resolution over previously used flow methods. Kinetics were measured both in the presence of the ADP analog and during steady-state hydrolysis of the ATP analog. These studies open the way for kinetic measurements during actin filament sliding on myosin tracks.

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“…In a thermally driven motor, no individual conformational change is required to account for all the work done. For example, the 4nm movement detected for an individual myosin molecule [9] may be regarded as a manifestation of the structural rearrangement that occurs within the motor domain to change its affinity to the track, rather than the stroke that carries out all of the work done; it may then be easier to understand how steps much longer than 4nm can be taken.…”

Section: Thermal Modelsmentioning

confidence: 99%

Molecular motors: not quite like clockwork

Amos

2008

Cell. Mol. Life Sci.

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Models commonly used to explain the mechanism of myosin motors typically include a powerstroke that is attributed to a conformational change in the motor domain and amplified by a long leverarm that connects the motor domain to the cargo. Similar models have proved less enlightening in the case of microtubule motors, for which it may be more helpful to consider models involving thermally driven mechanisms.

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Movement and force produced by a single myosin head

Cited by 592 publications

References 12 publications

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

Inorganic Phosphate Binds to the Empty Nucleotide Binding Pocket of Conventional Myosin II

Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis

Molecular motors: not quite like clockwork

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