Question: 

During its working stroke myosin proceeds through a series of structural states, driven by changes in the nucleotide. Details of the coupling between nucleotide state and myosin conformation are still unclear. It is, however, generally assumed that before the working stroke the myosin head has low affinity for actin and is in a pre-power stroke conformation with the lever arm in an up orientation. During the working stroke the myosin head is thought to change to a post-power stroke conformation with high actin affinity and the lever in a down orientation. An alternative possibility is that myosin can assume both, the pre- and post-power stroke conformation in any state of the nucleotide while the nucleotide only changes the relative occupancy of the two conformations. To address this point we aimed to see whether in the presence of ADP or without nucleotide, i.e., when myosin binds tightly to actin near the end of its working cycle, we can accumulate myosin heads in a closed, pre-power stroke state with the lever arm in an up orientation.

Methods: 

We identified myosin heads in a closed, pre-power stroke state by (1) prominent myosin based layer lines in 2D-X-ray diffraction patterns and by (2) rapidly reversible, low affinity binding to actin, revealed by fiber stiffness measurements. In the post-power stroke states with the lever arm in a down orientation, myosin based layer lines are weak, actin affinity is high, and dissociation from actin is very slow.

Results: 

Testing nucleotide analogs and small molecule effectors we found N-Benzyl-p-toluenesulfonamide (BTS) to accumulate myosin heads, both in the absence of nucleotide and in the presence of ADP, in structural states that generate strong myosin based layer lines. While BTS had no effect on myosin based layer lines in the presence of ATP, in the presence of ADP or without nucleotide BTS generated myosin based layer lines almost as strong as found in the presence of ATP at high temperature. This suggests that with BTS we could trap essentially all myosin heads in a closed, pre-power stroke conformation with the lever arm in an up orientation even in the absence of nucleotide. Measurements of fiber stiffness in the absence of nucleotide showed that nucleotide free myosin heads in the presence of BTS show rapidly reversible actin binding with much lower affinity for actin than seen in the absence of BTS.

Conclusions: 

While in the absence of nucleotide or in the presence of ADP myosin heads predominantly occupy post-power stroke states with high actin affinity, BTS apparently can trap myosin heads in a closed, pre-power stroke conformation even in the absence of nucleotide or with ADP in the active site. This is consistent with the concept that in all nucleotide states myosin can assume two conformations, a pre- and a post-power stroke conformation, and that the state of the nucleotide only modulates the relative occupancies, e.g., from mostly in the pre-power stroke conformation in weak binding states to almost all in the post-power stroke conformation in the presence of ADP or without nucleotide in the active site.