Characterizing the concentration and load dependence of phosphate binding to rabbit fast skeletal actomyosin.

Intensely contracting fast skeletal muscle rapidly loses the ability to generate force, due in part to the accumulation of phosphate (P) inhibiting myosin's force-generating capacity, in a process that is strain dependent. Crucial aspects of the mechanism underlying this inhibition remain unclear. Therefore, we directly determined the effects of increasing [P] on rabbit psoas muscle myosin's ability to generate force against progressively higher resistive loads in a laser trap assay, with the requisite spatial and temporal resolution to discern the mechanism of inhibition.

FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V.

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.