We have used saturation transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin heads in bundles of skinned muscle fibers, under conditions of rigor, relaxation, and isometric contraction. Experiments were performed on fiber bundles perfused continuously with an ATP-regenerating system. Conditions were identical to those we have used in previous studies of myosin head orientation, except that the fibers were perpendicular to the magnetic field, making the spectra primarily sensitive to rotational motion rather than to the orientational distribution. In rigor, the high intensity of the ST-EPR signal indicates the absence of microsecond rotational motion, showing that heads are all rigidly bound to actin. However, in both relaxation and contraction, considerable microsecond rotational motion is observed, implying that the previously reported orientational disorder under these conditions is dynamic, not static, on the microsecond time scale. The behavior in relaxation is essentially the same as that observed when myosin heads are detached from actin in the absence of ATP (Barnett and Thomas, 1984), corresponding to an effective rotational correlation time of approximately 10 microseconds. Slightly less mobility is observed during contraction. One possible interpretation is that in contraction all heads have the same mobility, corresponding to a correlation time of approximately 25 microseconds. Alternatively, more than one motional population may be present. For example, assuming that the spectrum in contraction is a linear combination of those in relaxation (mobile) and rigor (immobile), we obtained a good fit with a mole fraction of 78–88% of the heads in the mobile state.(ABSTRACT TRUNCATED AT 250 WORDS)
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We thank Piotr Fajer and Roger Cooke for helpful discussions; Robert L. H. Bennett and Kiandokt Beyzavi for technical assistance, and John Lipscomb for making the Varian E-109 spectrometer and computer available. We thank Piotr Fajer for mechanical measurements. This work was supported by grants from the National Institutes of Health (AR 32961 and RR03826) and the Muscular Dystrophy Asso- ciation of America. Vincent A. Barnett was partially supported by a predoctoral fellowship from the National Science Foundation. David D. Thomas was supported by an Established Investigatorship from the American Heart Association.