We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 μm (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 °), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread >90 °) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institutes of Health (GM 27906 and AM 32961). the American Heart Association (80-850), the Kational Science Foundation (PCM 8004612), and the Muscular Dystrophy Association of America. D.D.T. was supported by a Research Career Development Award from the National Institutes of Health, and is currently supported by an Established Investigatorship from the American Heart Association. V.A.B. is supported by a predoctoral fellowship from the National Science Foundation.