| The mechanism by which muscle generates force has been the subject of considerable study. Key to our understanding of this mechanism is the conformational changes occurring in the myosin “head” as it interacts with the thin filament. Each myosin head individually binds to the thin filament, hydrolyzes ATP and generates force. This study has used electron paramagnetic resonance (EPR) spectroscopy to study the structure and function of the regulatory domain located on the distal portion of the myosin head.; The mobility of the essential light chain (ELC) and the regulatory light chain (RLC) subdomains of the regulatory domain was individually measured utilizing saturation transfer electron paramagnetic resonance (ST-EPR) spectroscopy. Their mobilities were found to be similar, and that similarity persisted under conditions, which increase the overall mobility of the myosin head: upon RLC phosphorylation, an increase of pH or the presence of divalent cations. Modeling of this mobility enabled calculation of the persistence length of the regulatory domain, which at 1.5 μm, is adequate for it to serve as a lever arm. This is consistent with theories of force generation where the regulatory domain serves as a lever to amplify movement of the catalytic domain during the power stroke of striated muscle.; To analyze the orientation of the RLC of skeletal muscle fiber, a method to reconstitute skeletal muscle fiber with spin labeled skeletal RLC stoichiometrically, and with retention of full functionality was developed. The EPR spectra of muscle fiber, having exchanged RLC labeled with the spin label InVSL (at Cys-154) displayed two populations of RLC. One population consisted of isotropically disordered species, while the other was a highly ordered subpopulation. The ordering of that subpopulation is determined by actin binding; demonstrated by the decrease number of ordered heads during relaxation, contraction or in fibers stretched beyond overlap. |
