Myosin Crosslinking and EPR Capture the Start of Force Generation in Muscle Fibers

Abstract

Crosslinking the two most reactive Cys (SH1 and SH2) of the myosin catalytic domain (CD) inhibits force production and ATP hydrolysis and locks myosin in a weak actin-binding conformation with the CD immobilized and orientationally disordered. These results suggest that crosslinking traps a state in which the myosin head is on the cusp of force generation. In the present study, we measured the structural dynamics of myosin's light chain domain (LCD) in skeletal muscle fibers during rigor, relaxation, and with SH1 and SH2 crosslinked. To measure LCD structural dynamics, we exchanged spin labeled RLC for native RLC in permeabilized muscle fibers, with retention of function, then used EPR spectroscopy to measure structural dynamics. EPR spectra indicate when SH1 and SH2 are crosslinked, the LCD is in an orientation intermediate between relaxation and rigor, indicative of a state beginning to generate force. The saturation transfer EPR (STEPR) spectrum from these fibers does not change with crosslinking, demonstrating that that the LCD undergoes very slow dynamics, as in rigor, and is less dynamic than relaxation. In order to relate LCD structural dynamics with those of the CD, we measured CD structural dynamics in fibers by directly crosslinking SH1 and SH2 with BSL. EPR spectra from these fibers reveal that the CD is highly disordered, with dynamics ten times slower than in relaxation. Thus when SH1 and SH2 are crosslinked, both domains exhibit structural dynamics intermediate between relaxation (pre-power stroke) and rigor (post-power stroke). This supports the conclusion that SH1-SH2 crosslinking traps a state analogous to an initial force-generating state. We propose that this state is the missing link needed to explain how myosin undergoes a transition from dynamic disorder to order as it converts chemical energy to mechanical work.