Motion of Subfragment-1 in Myosin and Its Supramolecular Complexes: Saturation Transfer Electron Paramagnetic Resonance

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^-3 sec. Rotational correlation times (τ ₂) were determined for a spin label analog of iodoacetamide bound to the subfragment-1 (S-1) 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-1 (τ ₂ = 2 × 10^-7 sec) and can be used to estimate values of τ ₂ for the S-1 region of the myosin molecule; (b) in solutions of intact myosin, S-1 has considerable mobility relative to the rest of the myosin molecule, the value of τ ₂ for the S-1 segment of myosin being less than twice that for isolated S-1, while the molecular weights differ by a factor of 4 to 5; (c) in myosin filaments, τ ₂ increases by a factor of only about 10, suggesting motion of the S-1 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-1 increases τ ₂ by a factor of nearly 10³, indicating strong immobilization of S-1 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.