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

Abstract

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.ABSTRACT 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, cryoelectron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide highresolution 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 Ca 2+ , 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.

Atomistic Models from Orientation and Distance Constraints Using EPR of a Bifunctional Spin Label

“…Tight coupling of spin labels to the protein backbone was a goal that inspired the original synthesis of BSLs (5), and this property has since been well-established in the literature (7,9,(42)(43)(44)(45). In this work, we demonstrate that BSLs are not only strongly immobilized and well-ordered on a-helices but also highly stereoselective.…”

Electron tomography of muscle cross- bridge by regulatory light chain labelling with APEX2

“…Electron paramagnetic resonance (EPR) spectroscopy is an innovative method developed for mapping protein domain orientations in the cellular environment (Cooke et al, 1982, Barnett & Thomas, 1989, Zhao et al, 1996, Nogara et al, 2016, Savich et al, 2019. Moreover, this approach can complement high resolution in vitro methods like x-ray crystallography and NMR by providing structural information from protein domains in the native environment.…”