We have measured the rotational motion of myosin heads in synthetic thick filaments at 4 °C in the time range from 10-7 to 10 -4 seconds, by measuring transient absorption anisotropy of an eosin probe attached to a reactive sulfhydryl on the myosin head. Under conditions that result in monomeric myosin (500 mM ionic strength), the anisotropy decay is independent of pH in the range from 7.0 to 8.2 and [Mg2+]in the range from 0.1 to 10 mM; the anisotropy decays bi-exponentially with correlation times of 0.4 and 2 microseconds to a constant value of 0.016. Under more physiological conditions (115 mM ionic strength), resulting in filament formation, the anisotropy decay is sensitive to both pH and [Mg2+]. The anisotropy at pH 8.2 and 0.1 mM-Mg2+ decays with correlation times of 0.5 and 3.8 microseconds to a constant limiting anisotropy of 0.038. When the [Mg2+]is increased to 10 mM, the correlation times are 0.6 and 5.7 microseconds and the limiting anisotropy value is 0.055. Identical changes in the anisotropy decay are caused by an increase in [H+]to pH 7.0, in the presence of 0.1 mM-Mg2+. Increasing the total ionic strength to 187 mM decreases the amplitude of the cation effects. These results provide direct evidence that the rotational dynamics of myosin heads in thick filaments are influenced by physiological concentrations of cations. The results are qualitatively consistent with the proposal that these and other ionic conditions regulate transitions between "spread" and "compact" cross-bridge conformations, but the quantitative results indicate that cross-bridges undergo large-amplitude microsecond rotations even under conditions where the compact state should predominate.
Bibliographical noteFunding Information:
The instrument used in this work was designtd. constzucted and maintainrd primaril?-by Robert, 1.. H Bennett. following initial work by Brian P. C’itak. (‘omputer programming assistance was provided 11~ Kerry M. Lindahl. Joel T. Schneider and .Joseph A. Stone. Labeled myosin samples were prt~pared and characterized with the assistance of JIavitf A. Moment This work was supported by prant)s from t,he Xational Institutes of Health ((:,21 279Oti. AMXBtil and ILR01439). the American Heart Association (80-850). thts Sational Science Foundation (WM8004612). and tht Muscular Dystrophy Association of America. I). I1.T. was supported by an JCstablishrd fnvestigat,orship fiwm the American Heart Association.