Myosin motors that cannot bind actin leave their folded OFF state on activation of skeletal muscle.

Abstract

The myosin motors in resting skeletal muscle are folded back against their tails in the thick filament in a conformation that makes them unavailable for binding to actin. When muscles are activated, calcium binding to troponin leads to a rapid change in the structure of the actin-containing thin filaments that uncovers the myosin binding sites on actin. Almost as quickly, myosin motors leave the folded state and move away from the surface of the thick filament. To test whether motor unfolding is triggered by the availability of nearby actin binding sites, we measured changes in the x-ray reflections that report motor conformation when muscles are activated at longer sarcomere length, so that part of the thick filaments no longer overlaps with thin filaments. We found that the intensity of the M3 reflection from the axial repeat of the motors along the thick filaments declines almost linearly with increasing sarcomere length up to 2.8 µm, as expected if motors in the nonoverlap zone had left the folded state and become relatively disordered. In a recent article in JGP, Squire and Knupp challenged this interpretation of the data. We show here that their analysis is based on an incorrect assumption about how the interference subpeaks of the M3 reflection were reported in our previous paper. We extend previous models of mass distribution along the filaments to show that the sarcomere length dependence of the M3 reflection is consistent with <10% of no-overlap motors remaining in the folded conformation during active contraction, confirming our previous conclusion that unfolding of myosin motors on muscle activation is not due to the availability of local actin binding sites.