Structural Dynamics Of Myosin'S Light Chain Domain In A Pre-power Stroke Conformation

Abstract

Cross-linking the two most reactive Cys of the myosin catalytic domain (CD) (SH1 and SH2) inhibits force production and ATP hydrolysis and locks myosin in a weak actin-binding conformation. Recent work using a bifunctional spin label (BSL) to crosslink SH1 and SH2 has shown that the CD is immobilized and orientationally disordered, suggesting that cross-linking traps myosin in a an intermediate state that primes the myosin head to generate force (Thompson et al., BJ, in press). In the present study, we measured light chain domain (LCD) structural dynamics in muscle fibers as a function of SH crosslinking. If the CD really is orientationally disordered by crosslinking and the two domains are structurally coupled, some of this disorder should be propagated to the LCD. To measure LCD structural dynamics, we used site-directed spin labeling to label chicken gizzard regulatory light chain (RLC), and then exchanged this labeled RLC for the native RLC in rabbit psoas fibers. Prior to crosslinking, EPR spectra acquired with the fiber axis parallel and perpendicular to the external field were very different, but cross-linking decreases this difference, indicating increased disorder. Saturation transfer EPR on these fibers showed that the heads remained immobile and thus attached to actin. These results support our hypothesis that SH1-SH2 crosslinking traps an actomyosin complex, possibly the first force-generating state in the power stroke, in which the CD is highly disordered and LCD is partially disordered, indicating a partially flexible linkage. A secondary goal of this research is to improve the technology for EPR on muscle fibers by developing a novel high-sensitivity EPR resonator for analysis of spin-label mobility, orientation, and force on labeled fibers.