A mutation in switch I alters the load-dependent kinetics of myosin Va.

Myosin Va is the molecular motor that drives intracellular vesicular transport, powered by the transduction of chemical energy from ATP into mechanical work. The coupling of the powerstroke and phosphate (P_i) release is key to understanding the transduction process, and crucial details of this process remain unclear. Therefore, we determined the effect of elevated P_i on the force-generating capacity of a mini-ensemble of myosin Va S1 (WT) in a laser trap assay. By increasing the stiffness of the laser trap we determined the effect of increasing resistive loads on the rate of P_i-induced detachment from actin, and quantified this effect using the Bell approximation. We observed that WT myosin generated higher forces and larger displacements at the higher laser trap stiffnesses in the presence of 30 mM P_i, but binding event lifetimes decreased dramatically, which is most consistent with the powerstroke preceding the release of P_i from the active site. Repeating these experiments using a construct with a mutation in switch I of the active site (S217A) caused a seven-fold increase in the load-dependence of the P_i-induced detachment rate, suggesting that the S217A region of switch I may help mediate the load-dependence of P_i-rebinding. Myosin transduces chemical energy into mechanical work, but the mechanism remains unclear. In this work, the authors show that force-generation precedes product release and that a mutation in the active site alters the load dependence of product release. [ABSTRACT FROM AUTHOR]