| Abstract
| - We have used electron paramagnetic resonance (EPR) of spin-labeled scallop muscle, inconjunction with laser flash photolysis of caged ATP, to resolve millisecond rotational transitions of themyosin light-chain domain (LCD) during transient force generation. We previously used EPR to resolvetwo 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 structuralstates with biochemical states in the actin−myosin ATPase reaction, and showed that a small shift in thesteady-state distribution between these two LCD orientations (i.e., a net lever arm rotation) is associatedwith force generation in muscle. In the study presented here, we measured millisecond changes in thisorientational distribution (i.e., the rates of transition between the two LCD orientations) in muscle followingflash photolysis of caged ATP, in both the presence and absence of Ca. The transient acquired in theabsence of Ca is dominated by a rapid (1/τ1 = 37 s-1) disordering transition from the single orientationin rigor to the bimodal orientation distribution observed for detached cross-bridges in relaxation (i.e., thereversal of the lever arm rotation), followed by a recovery phase (1/τ2 = 2.4 s-1) of very small amplitude(small fraction of heads participating). In the presence of Ca, the transient exhibited a similar initialdisordering phase (1/τ1 = 38.5 s-1), followed by a recovery phase (1/τ2 = 8.33 s-1) of substantial amplitude,corresponding to the forward rotation and ordering of the lever arm. A standard kinetic model was usedto 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 forcedevelopment (91 s-1) is faster than the rate of the forward lever arm rotation (8 s-1). This observedrelationship between the kinetics of the lever arm rotation and transient force development in muscleprovides new insight into how myosin both generates and responds to muscle force.
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