Direct observation of phosphate inhibiting the force generating capacity of myosin using a laser trap assay

The accumulation of phosphate (Pi) is believed to cause much of the loss in the force during fatigue, but the molecular basis of this effect remains poorly understood. To gain insight into this process we determined the effect of a fatiguing level of Pi (30mM) on the force‐generating capacity of a mini‐ensemble of skeletal myosin (~8molecules) interacting with a single actin filament using a three bead laser trap assay, at 100uM ATP. In the absence of Pi the average force generated displayed a bimodal Gaussian distribution with a low force peak centered at 0.30 ± 0.02 pN and second higher peak centered at 1.22 ± 0.08 pN. In the presence of Pi a low force peak centered at 0.28 ± 0.02 pN remained but the high force peak was completely absent, greatly reducing the average force produced. The duration of the low force binding events were statistically similar in both the presence (30 ± 4ms) and absence of Pi (28 ± 3ms) as were the average displacements of these events at 14 ± 1nm and 16 ± 1nm respectively, consistent with a mix of single and double molecule binding events. The high force events generated on average 64 ± 4nm displacements lasting 75 ± 8ms, suggesting these resulted from multiple binding events before detachment from the actin filament. The overall frequency of binding events was similar in the presence (3.0/s) and absence of Pi (2.7s). Taken together these data suggest that Pi reduces the force‐generating capacity of a myosin by eliminating the ability to make the successive and simultaneous actin attachments required to generate higher forces.