Transient kinetics and mechanics of myosin's force-generating rotation in muscle: resolution of millisecond rotational transitions in the spin-labeled myosin light-chain domain.

摘要

We have used electron paramagnetic resonance (EPR) of spin-labeled scallop muscle, in conjunction with laser flash photolysis of caged ATP, to resolve millisecond rotational transitions of the myosin light-chain domain (LCD) during transient force generation. We previously used EPR to resolve two distinct orientations of the LCD [Baker, J. E., Brust-Mascher, I., Ramachandran, S., LaConte, L. E., and Thomas, D. D. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2944-2949], correlated these structural states with biochemical states in the actin-myosin ATPase reaction, and showed that a small shift in the steady-state distribution between these two LCD orientations (i.e., a net lever arm rotation) is associated with force generation in muscle. In the study presented here, we measured millisecond changes in this orientational distribution (i.e., the rates of transition between the two LCD orientations) in muscle following flash photolysis of caged ATP, in both the presence and absence of Ca. The transient acquired in the absence of Ca is dominated by a rapid (1/tau(1) = 37 s(-1)) disordering transition from the single orientation in rigor to the bimodal orientation distribution observed for detached cross-bridges in relaxation (i.e., the reversal of the lever arm rotation), followed by a recovery phase (1/tau(2) = 2.4 s(-1)) of very small amplitude (small fraction of heads participating). In the presence of Ca, the transient exhibited a similar initial disordering phase (1/tau(1) = 38.5 s(-1)), followed by a recovery phase (1/tau(2) = 8.33 s(-1)) of substantial amplitude, corresponding to the forward rotation and ordering of the lever arm. A standard kinetic model was used to fit these data, revealing rate constants consistent with those previously determined by other methods. Surprisingly, a comparison of the EPR transients with force transients reveals that the rate of force development (91 s(-1)) is faster than the rate of the forward lever arm rotation (8 s(-1)). This observed relationship between the kinetics of the lever arm rotation and transient force development in muscle provides new insight into how myosin both generates and responds to muscle force.