Structural dynamics of the actomyosin complex probed by a bifunctional spin label that cross-links SH1 and SH2

Andrew R. Thompson, Nariman Naber, Clyde Wilson, Roger Cooke, David D. Thomas

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We have used a bifunctional spin label (BSL) to cross-link Cys 707 (SH1) and Cys697 (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.

Original languageEnglish (US)
Pages (from-to)5238-5246
Number of pages9
JournalBiophysical journal
Issue number11
StatePublished - Dec 1 2008

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health (NIH) grants AR032961 (D.D.T.) and AR42895 (R.C.). A.R.T. was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases/NIH training grant AR007612. We thank Leanne Kolb, Christina Yi, Sarah Blakely, and Octavian Cornea for excellent technical support; Yuri Nesmelov, Roman Agafonov, Ryan Mello, Jenny Klein, Ewa Prochniewicz, and Jack Surek for insightful discussions; and Edmund Howard for EPR analysis software.


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