Helix-coil melting in rigor and activated cross-bridges of skeletal muscle.

The studies described in this paper focus on the structural stability of the S-2 segment of the myosin cross-bridge in rigor, relaxed and activated muscle. Enzyme-probe observations of myofibrils of rabbit psoas muscle in rigor reveal that the alpha-helical LMM/HMM hinge domain of S-2 undergoes substantial local melting near physiological temperatures when the S-2 portion of the cross-bridge is detached from the thick filament surface. This process is strongly suppressed under ionic conditions where the cross-bridge is bound to the filament backbone. Activation of glycerinated fiber bundles results in a dramatic increase (approximately 100 fold compared to rigor and relaxed fibers) in the rate of chymotryptic cleavage in the hinge domain consistent with an increase in local melting at several sites encompassing this region. Comparative plots of the apparent rate-constant for cleavage within the S-2 hinge and the isometric force generated by active fibers versus [MgATP] give similar profiles suggesting a close coupling between this conformational transition and contractile force. This interpretation appears to be in accord with recent laser T-jump experiments of rigor ("bridges up") and activated psoas muscle fibers which also suggest coupling between melting in S-2 and force generation.