Biophysical Society Thematic Meeting | Canterbury 2023

Towards a More Perfect Union: Multi-Scale Models of Muscle and Their Experimental Validation

Monday Speaker Abstracts

RECENT BIOPHYSICAL APPROACHES TO INVESTIGATE THE COUPLING BETWEEN FORCE-GENERATION AND PHOSPHATE-RELEASE IN MYOSIN Edward P. Debold 1 University of Massachusetts, Amherst, Kinesiology, Amherst, MA, USA Myosin is the molecular motor that generates the force and motion required to contract our muscles and drive a host of intracellular processes. It does so by transducing chemical energy, derived from the hydrolysis of ATP, into mechanical work. Despite extensive study key aspects of the transduction process remain unclear, most notably the nature of the coupling between the force-generating powerstroke and the release of phosphate (P i ) from the active site. We have performed a series of biochemical and biophysical studies aimed at gaining a deeper understanding of this coupling, including determining the relative timing of the powerstroke and P i -release. Transient kinetic and FRET-based experiments on myosin Va indicate that the powerstroke occurs more than twice as fast as the rate of P i -release. Single molecule laser trap assays, using both myosin II and myosin Va, demonstrate that the powerstroke is generated rapidly (<2ms) after the formation of a strong actomyosin bond. Indeed, even in construct with a mutation (S217A) designed to slow P i -release from the active site myosin Va generates a powerstroke a few milliseconds after strongly binding to actin. These findings are most consistent with the powerstroke preceding the release of P i from the active site. We have also examined how a resistive load affects myosin’s rate of P i -induced detachment from actin. Findings from myosin Va and myosin II indicate that this detachment rate is highly load sensitive, and that a mutation in the switch I region of myosin’s active site dramatically alters the load dependence of this rate. Thus, these new finding are providing novel insights into the nature of energy transduction by myosin, which we hope will lead to improved models of muscle contraction and myosin-driven intracellular processes.

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