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 i ) 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 i in its active site, by either elevating P i in solution or by introducing a mutation in myosin's active site (S217A) to slow P irelease from the active site. Upon binding to the actin filament, WT myosin generated a powerstoke rapidly (≥500 s À1 ) and without a detectable delay, both in the absence and presence of 30 mM P i . The elevated levels of P i did, however, affect event lifetime, eliminating the longest 25% of binding events, confirming that P i 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 i release rate. These findings provide direct evidence that myosin Va generates a powerstroke with P i still in its active site.

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force generating lever arm in myosin V. We directly compared the FRET results with single molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), while our simulations suggest the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre- and post-power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

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Myosin is a molecular motor responsible for generating the force and/or motion that drive many intracellular processes, from muscle contraction to vesicular transport. It is powered by its ability to convert the chemical energy, released from the hydrolysis of ATP, into mechanical work. The key event in the transduction process is the coupling of the force-generating powerstroke with the release of phosphate (Pi) from the active site, but the mechanisms and the structural elements involved in this coupling remain unclear. Therefore, we determined the effect of elevated levels of Pi on the force-generating capacity of a mini-ensemble of myosin Va molecules (WT) in a three-bead laser trap assay. We quantified the load-dependence of the Pi-induced detachment rate by performing the experiments at three different laser trap stiffnesses (0.04, 0.06 and 0.10pN/nm). Myosin generated higher peak forces at the higher laser trap stiffnesses, and the distance the myosin displaced the actin filament significantly increased in the presence of 30mM Pi, a finding most consistent with the powerstroke preceding Pi-release. In contrast, the duration of the binding events was significantly reduced at higher trap stiffness in the presence of Pi, indicating that the higher resistive force accelerated the rate of Pi-induced detachment from actin. A Bell approximation, was used to quantify the load-dependence of this rate (k1 = ko x exp(Fd/kt)), revealing a d-value of 0.7nm for the WT myosin. Repeating these experiments using a construct with a mutation (S217A) in a key region (Switch I) of the nucleotide-binding site increased myosin’s sensitivity to load five-fold (d = 3.5nm). Thus, these findings provide a quantitative measure of the force-dependent nature of Pi-rebinding to myosin’s active site and suggest that this effect involves the switch I element of the nucleotide-binding pocket. These findings, therefore, provide important new insights into the mechanisms through which this prototypical motor enzyme couples the release of chemical energy to the generation of force and/or motion.

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