Conformational selection during weak binding at the actin and myosin interface

The molecular mechanism of the powerstroke in muscle is examined by resonan ce energy transfer techniques. Recent models suggesting a pre-cocking of th e myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the regulatory light chain in the presence of nucleotide transition state analogs. Only small changes (<0.5 n m) were detected that are consistent with internal conformational changes o f the myosin molecule, but not with extreme differences in the average leve r arm position suggested by some atomic models. These results were confirme d by stopped-flow resonance energy transfer measurements during single ATP turnovers on myosin, To examine the participation of actin in the powerstro ke process, resonance energy transfer between the regulatory light chain on myosin subfragment-1 and the C-terminus of actin was measured in the prese nce of nucleotide transition state analogs. The efficiency of energy transf er was much greater in the presence of ADP-AlF4, ADP-BeFx, and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound d ifferences in the conformations of the weakly and conformational selection that occurs during the weak binding strongly attached cross-bridges that ap pear to result from a of the myosin head to actin.