The cyclic interaction of myosin and actin are at the heart of the mechanism of muscle contraction and cell motility. A model of the molecular mechanism of muscle contraction has not been definitively confirmed. The purpose of the proposed experiments is to elucidate this mechanism by detailed studies of protein orientation and conformation. The interaction of myosin and actin will be investigated using static and time resolved techniques developed for fluorescence and electron spin resonance (ESR) probes. These probes will be covalently and specifically attached primarily to two myosin sulfhydryl side chains in muscle fibers, either SH1 or SH2. The probes will report their orientation as a function of time and physiological state of the fiber. We will use novel ESR and fluorescence techniques to investigate the static and time resolved angular distribution of probes attached to the components of the contractile apparatus. In the static experiments, we investigate the orientation of the cross-bridge using fluorescence polarization spectroscopy. With FPS the orientation of the transition dipole of the probe rotates on the cross-bridge, due to excitation wavelength variation, allowing a more general study of the cross-bridge angular degrees of freedom. With ESR the probe angular distribution is reconstructed, with high angular resolution, from a series of ESR spectra originating from a muscle fiber at differing tilt angles relative to the Zeeman magnetic field. The fluorescence polarization and ESR spectra will be measured from labeled muscle fibers in a variety of physiological states to determine the cross-bridge orientation in these states and to correlate this data with the models for cross-bridge participation in muscle contraction. In the time-resolved experiments, we will investigate the rotational motions of cross-bridges during muscle contraction on the time domain of 10-6 to 10+3 seconds. This will be done using the technique of polarized fluorescence photobleaching recovery (PFPR) applied to labeled muscle fibers. With PFPR a brief pulse of polarized focused light irreversibly photobleaches probes with transition dipoles aligned with the polarization of the light in the focused spot. The bleached region is monitored with an attenuated polarized light source and the recovery of fluorescence is observed as unbleached fluorophores rotate and their dipoles move into alignment with the attenuated polarized source.
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