Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Elevated levels of phosphate (P-i) reduce isometric force, providing support for the notion that the release of P-i from myosin is closely associated with the generation of muscular force. P-i is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of P-i on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of P-i on the force-generating capacity of a small ensemble of myosin (similar to 12 myosin heads) using a three-bead laser trap assay. In the absence of P-i, myosin pulled the actin filament out of the laser trap an average distance of 54 +/- 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM P-i, myosin generated only enough force to displace the actin filament by 13 +/- 1 nm, generating just 0.2 pN of force. The elevated P-i also caused a >65% reduction in binding-event lifetime, suggesting that P-i induces premature detachment from a strongly bound state. Definitive evidence of a P-i-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which P-i induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of P-i on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that P-i-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.