Abstract
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. 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 (±15º) with the spin-label z axis parallel to actin, and a second population with a large distribution (±60 º). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than with 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. Comparison with electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 º of the electron microscopy model.
In smooth muscle, force generation is regulated by phosphorylation of RLC, which is thought to change the relative positions of myosin heads. To verify this hypothesis, we have measured the distance between selected cysteine mutants of RLC in 6S myosin monomer by conventional and pulse EPR. In the unphosphorylated smooth muscle myosin (SMM) monomer, the distances were determined to be 43% of all RLC sites with distance of 30 Å for SMM Q15C; 36% of 14.7 Å and 22% of 39 Å for SMM N38C; 38% of 30 Å and 21% of 48 Å for SMM S59C; 43% of 35 Å and 33% of 48 Å for SMM T96C; 37% of 33 Å for SMM C108. These results implied that the two RLCs were parallel to each other, leaving the two N domains in close proximity and two C domains far apart. Phosphorylation increased the head-head distance of about 30% SMM N38C and SMM S59C to beyond the limit of EPR measurement. Surprisingly, the distance of two RLCs T96C was still in the range of 18-60 Å. Not only had phosphorylation increased the head-head distance, the width of distance was observed to increase in SMM S59C and T96C. When phosphorylated the rigid conformer of SMM completely vanished and all SMMs become very flexible.
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