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Myosin Orientation in a Muscle Fiber Determined with High Angular Resolution Using Bifunctional Spin Labels

We have measured the orientation of the myosin light chain domain in demembranated muscle fibers by electron paramagnetic resonance (EPR), using a bifunctional spin label (BSL), with angular resolution of 4 degrees. Despite advances in X-ray crystallography, cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain under ambient conditions in fiber. Two cysteines, 4 residues apart, were engineered on an α helix in the myosin regulatory light chain (RLC), permitting stereoselective site-directed labeling with BSL. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtained EPR spectra that resolved angular distributions in several biochemical states. The accurate determination of BSL’s angular coordinates allowed us to determine the orientation of individual structural elements with respect to the muscle fiber axis. The addition of ATP, in the absence of Ca2+, shifted the orientation to a much more disordered distribution. We resolved an oriented population with lever arm oriented perpendicular to the muscle fiber axis, a state previously observed directly in insect flight muscle, but not in fibers having isolated myosin heads (S1) attached to actin. Summary We used electron paramagnetic resonance to determine the orientation of elements within the myosin regulatory light chain in skinned skeletal muscle fibers. A bifunctional spin label provided sufficient resolution to detect an ordered population of lever arms perpendicular to actin.