Rotational dynamics of actin-bound intermediates in the myosin ATPase cycle.
We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin subfragment one (MSL-S1) bound to actin in the presence of the ATP analogues AMPPNP (5'-adenylylimido diphosphate) and ATP gamma S [adenosine 5'-O-(3-thiotriphosphate)], which are believed to trap myosin in strongly and weakly bound intermediate states of the actomyosin ATPase cycle, respectively. Sedimentation binding measurements were used to determine the fraction of myosin heads bound to actin under ST-EPR conditions and the fraction of heads containing bound nucleotide. ST-EPR spectra were then corrected to obtain the spectrum corresponding to the ternary complex (actin.MSL-S1.nucleotide). The ST-EPR spectrum of MSL-S1.AMPPNP bound to actin is identical to that obtained in the absence of nucleotide (rigor complex), indicating no rotational motion of MSL-S1 relative to actin on the microsecond time scale. However, MSL-S1-ATP gamma S bound to actin is rotationally mobile, with an effective rotational correlation time (tau r) of 17 +/- 2 microseconds. This motion is similar to that observed previously for actin-bound MSL-S1 during the steady-state hydrolysis of ATP [Berger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8753-8757]. We conclude that, in solution, the weakly bound actin-attached states of the myosin ATPase cycle undergo microsecond rotational motions, while the strongly bound intermediates do not, and that these motions are likely to be involved in the molecular mechanism of muscle contraction.
Berger
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CL
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Thomas
D D
DD
D000199:Actins / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels; D004578:Electron Spin Resonance Spectroscopy; D009218:Myosins / Q000737:chemistry*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
D000199:Actins; D000345:Affinity Labels; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D009218:Myosins