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Myosin Regulatory Domain Orientation in Skeletal Muscle Fibers: Application of Novel Electron Paramagnetic Resonance Spectral Decomposition and Molecular Modeling Methods

Bruce A. J. Baumann ^* , Hua Liang  , Ken Sale  , Brett D. Hambly  and Piotr G. Fajer ^*  

^* Molecular Biophysics Graduate Program, The National High Magnetic Field Laboratory, Department of Biological Sciences, Florida State University, Tallahassee, Florida; and Department of Pathology, University of Sydney, New South Wales, Australia

Correspondence: Address reprint requests to Piotr G. Fajer, Tel.: 850-644-2600; Fax: 850-644-1366, E-mail: fajer{at}magnet.fsu.edu.

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (±15°) with the spin-label z axis parallel to actin, and a second population with a large distribution (±60°). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10° of the electron microscopy model.

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