Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2.
We have used a bifunctional spin label (BSL) to cross-link Cys(707) (SH1) and Cys(697) (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the same weakened ATPase activity and actin-binding affinity that is observed when SH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state A.M.ADP.P. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1. When bound to actin in oriented muscle fibers in the absence of ATP, BSL-S1 showed almost complete orientational disorder, as reported previously for the weakly bound A.M.ADP. In contrast, helical order is observed for the strongly bound state A.M. Saturation transfer electron paramagnetic resonance showed that the disorder of cross-linked S1 on actin is nearly static on the microsecond timescale, at least 30 times slower than that of A.M.ADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of A.M.ADP, slow rotational mobility comparable to that of A.M, and intermediate actin affinity. These results support the hypothesis that the catalytic domain of myosin is orientationally disordered on actin in a post-hydrolysis state in the early stages of force generation.
Thompson
Andrew R
AR
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Naber
Nariman
N
Wilson
Clyde
C
Cooke
Roger
R
Thomas
David D
DD
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology*; D003545:Cysteine / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012399:Rotation; D013113:Spin Labels; D013379:Substrate Specificity; D018909:src Homology Domains
D000199:Actins; D003432:Cross-Linking Reagents; D015879:Myosin Subfragments; D013113:Spin Labels; D000255:Adenosine Triphosphate; D000205:Actomyosin; D003545:Cysteine