Myosin light-chain domain rotates upon muscle activation but not ATP hydrolysis.
We have studied the correlation between myosin structure, myosin biochemistry, and muscle force. Two distinct orientations of the myosin light-chain domain were previously resolved using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled regulatory light chains in scallop muscle fibers. In the present study, we measured isometric force during EPR spectral acquisition, in order to define how these two light-chain domain orientations are coupled to force and the myosin ATPase cycle. When muscle fibers are partially activated with increasing amounts of calcium, the distribution between the two light-chain domain orientations shifts toward the one associated with strong actin binding. This shift in distribution is linearly related to the increase in force, suggesting that rotation of the light-chain domain is coupled to strong actin binding. However, when nucleotide analogues are used to trap myosin in the pre- and posthydrolysis states of its ATPase cycle in relaxed muscle, there is no change in the distribution between light-chain domain orientations, showing that the rotation of the light-chain domain is not directly coupled to the ATP hydrolysis step. Instead, it is likely that in relaxed muscle the myosin thick filament stabilizes two light-chain domain orientations that are independent of the nucleotide analogue bound at the active site. We conclude that a large and distinct rotation of the light-chain domain of myosin is responsible for force generation and is coupled to strong actin binding but is not coupled to a specific step in the myosin ATPase reaction.
Brust-Mascher
I
I
Department of Biochemistry, Molecular Biology, & Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
LaConte
L E
LE
Baker
J E
JE
Thomas
D D
DD
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D004578:Electron Spin Resonance Spectroscopy; D006868:Hydrolysis; D009130:Muscle, Smooth / Q000502:physiology*; D018994:Myosin Light Chains / Q000737:chemistry* / Q000378:metabolism
D018994:Myosin Light Chains; D000255:Adenosine Triphosphate; D002118:Calcium