Molecular dynamics in spin-labeled muscle proteins was studied with a recently developed electron paramagnetic resonance (EPR) technique, saturation transfer spectroscopy, which is uniquely sensitive to rotational motion in the range of 10-7-10-sec. Rotational correlation times (r2) were determined for a spin label analog of iodoacetamide bound to the subfragment-I (S-i) region of myosin under a variety of conditions likely to shed light on the molecular mechanism of muscle contraction. Results show that (a) the spin labels are rigidly bound to the isolated S-i (i-2 = 2 X 10-7 sec) and can be used to estimate values of r2 for the S-i region of the myosin molecule; (b) in solutions of intact myosin, S-i has considerable mobility relative to the rest of the myosin molecule, the value of r2 for the S-i segment of myosin being less than twice that for isolated S-i, while the molecular weights differ by a factor of 4 to 5; (c) in myosin filaments, T2 increases by a factor of only about 10, suggesting motion of the S-i regions independent of the backbone of the myosin filament, but slower than that in a single molecule; (d) addition of F-actin to solutions of myosin or S-i increases T2 by a factor of nearly 103, indicating strong immobilization of S-i upon binding to actin. Saturation transfer spectroscopy promises to provide an extremely useful tool for the study of the motions of the crossbridges and thin filaments in reconstituted systems and in glycerinated muscle fibers.Current models of muscle contraction involve motion of the crossbridges between thick and thin filaments (1, 2). These crossbridges correspond to the subfragment-1 (S-1) and subfragment-2 (S-2) regions of myosin (3). Evidence for segmental flexibility within the myosin molecule, characterized by rotational correlation times (T2) on the order of 10-i sec, has been obtained by observing the rate of depolarization of fluorescent probes attached to the S-1 region of myosin in solution (4). Studies on the fluorescence polarization of tryptophyl residues in glycerinated muscle fibers suggest that the orientation of crossbridges may depend on the functional state, namely, contraction, relaxation, or rigor (5). However, there are no reliable measurements on the rates of S-1 motion in thick filaments or in actomyosin complexes.Some One of these techniques has proven applicable to the study of solutions of spin-labeled proteins: absorption EPR, detected 900 out-of-phase with respect to the unsaturated second harmonic EPR response to the modulation field, Hm cos Wmt (12, 13, 15). If T2 1wm A ' T71 --10-5 sec, and the spin system is partially saturated, the competition between spectral diffusion and field modulation, in governing the passage of spins through the resonance condition, gives rise to a significant signal component that lags behind the modulation by 900 and is sensitive in shape to rotational diffusion.In the present study we have used the saturation transfer spectra of a spin label rigidly attached to the S-1 region of myosin to study the...
