Structural Dynamics of the Actomyosin Complex Probed by a Bifunctional Spin Label that Cross-Links SH1 and SH2

ABSTRACT We have used a bifunctional spin label (BSL) to cross-link Cys707 (SH1) and Cys697 (SH2) in the catalytic domain of myosin subfragment 1 (S1). BSL induces the sameweakenedATPaseactivity andactin-binding affinity that is observedwhenSH1 and SH2 are cross-linked with pPDM, which traps an analog of the post-hydrolysis state AMADPP. Electron paramagnetic resonance showed that BSL reports the global orientation and dynamics of S1.When bound to actin in orientedmuscle fibers in the absence of ATP, BSL-S1 showedalmost complete orientational disorder, as reported previously for theweakly boundAMADP. In contrast, helical order is observed for the strongly bound state AM. 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 AMADP. We conclude that cross-linked S1 exhibits rotational disorder comparable to that of AMADP, slow rotational mobility comparable to that of AM, 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