Myosin's powerstroke occurs prior to the release of phosphate from the active site.

Myosins are a family of motor proteins responsible for various forms of cellular motility, including muscle contraction and vesicular transport. The most fundamental aspect of myosin is its ability to transduce the chemical energy from the hydrolysis of ATP into mechanical work, in the form of force and/or motion. A key unanswered question of the transduction process is the timing of the force-generating powerstroke relative to the release of phosphate (P ) from the active site. We examined the ability of single-headed myosin Va to generate a powerstroke in a single molecule laser trap assay while maintaining P in its active site, by either elevating P in solution or by introducing a mutation in myosin's active site (S217A) to slow P -release from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s ) and without a detectable delay, both in the absence and presence of 30 mM P . The elevated levels of P did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that P rebound to myosin's active site and accelerated detachment. The S217A construct also generated a powerstroke similar in size and rate upon binding to actin despite the slower P release rate. These findings provide direct evidence that myosin Va generates a powerstroke with P still in its active site. Therefore, the findings are most consistent with a model in which the powerstroke occurs prior to the release of P from the active site.