Contractions of muscle cells and the motility of nonmuscle cells are known to result from the interaction of myosin, actin and ATP. The long-range goal of our research is to eluidate the molecular mechanisms involved in these interactions. Once hypothetical mechanism envisions a cyclis interaction in which a myosin head attaches to an actin filament, and then rotates while attached to the filament thus pulling the filament towards the center of the sarcomere. Paramagnetic probes are excellent tools for investigating the contractile mechanism because both their angular distribution and their motion can be measured from their spectra. Our previous work with probes attached to a reactive sulfhydryl on the myosin head has questioned some aspects of the above conventional hypothesis by showing that at least one portion of the myosin head does not rotate during isometric force generation. We propose to extend these results in several directions. The possibility that myosin conformtion may be altered by filament motion will be investigated by obtaining spectra during slow fiber contractions and stretches. We will vary the isometric tension generated by the fibers and correlate tension with spectral changes to further isolate and quantify spectral components associated with myosin heads that are generating tension. Our previous data on contracting fibers was obtained using rabbit muscle and we will extend our results to frog muscle, smooth muscle and insect flight muscle to explore the generality of our findings. The results described above report on the orientation of the portion of myosin in the vicinity of the reactive sulfhydryl. Information from other probe sites is essential to understanding myosin motions during contraction, and a variety of sites will be explored. Preliminary experiments have found two possibilities for new probe sites: spin-labeled nucleotides bound to myosin and spin-labeled myosin light chains. When considered together the spectral data obtained at several sites under a variety of conditions will provide a more detailed picture of the changes in myosin orientation that lead to force production. These studies will be extended to elucidate how protein phosphorylation controls the contraction of skeletal and cardiac muscle. Knowledge of how force is produced and how it is controlled in skeletal muscle, and especially in cardiac muscle, will help define more rational therapies for treating disorders in these muscles.
