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

Abstract

ABSTRACTWe have measured the orientation of the myosin light chain domain (lever arm) elements 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 and 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 a muscle fiber. Two cysteines, 4 residues apart, were engineered on two α-helices in the myosin regulatory light chain (RLC), permitting stereoselective site-directed labeling with BSL. One labeled helix (helix E) is adjacent to the myosin lever arm, the other helix (helix B) is located farther apart from the motor domain beyond the “hinge” of the myosin. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtained EPR spectra that determined angular distributions of BSL with high resolution, which enabled the accurate determination of helix orientation of individual structural elements with respect to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibited both ordered (σ ~ 9-11 degrees) and disordered (σ > 38 degrees) populations. We used these angles to determine the orientation of the myosin lever arm, concluding that the oriented population has lever arms that are perpendicular to the muscle fiber axis. This orientation is ~33 degrees different than predicted from a standard “lever arm down” model based on cryo-EM of actin decorated with isolated myosin heads, but it is compatible with fluorescence polarization and EM data obtained from muscle fibers. The addition of ATP, in the absence of Ca2+, shifted the orientation to a much more disordered distribution.SummaryWe 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.