Relgulatory Light Chain Orientaiton on Myosin S1 Using a Bifunctional Spin Label

Abstract

We performed electron paramagnetic resonance (EPR) on the myosin head (S1), labeling the regulatory light chain (RLC) with a bifunctional spin label (BSL). To achieve stereoselective site-directed labeling with BSL, we engineered a pair of cysteines (i, i+4) in RLC. By exchanging BSL-labeled RLC onto S1 and decorating oriented muscle fibers with BSL-RLC-S1, we obtained EPR spectra from which the angular distribution of BSL can be determined with high resolution relative to the muscle fiber axis. In our previous work (Savich et al., JGP, 2019), we exchanged BSL-RLC on intact myosin in skinned muscle fibers and observed substantial angular disorder, which varied with the RLC labeling site. In the present study, papain-digested S1 spin-labeled with BSL and bound to actin in oriented muscle fibers, yields a single oriented population of helix E on RLC, with an angular dispersion of ∼10 degrees. Addition of ATP releases decorated myosin, showing the ATP dependence of actin binding is not perturbed by labeling. Activity of labeled S1 was verified by the actin-activated NADH ATPase assay. We conclude that the lack of uniform orientation in labeled RLC, observed previously in muscle fibers by both EPR and fluorescence, is due to strain between two myosin heads. Sensitivity of the spectral features to the flow rate presents a method to measure a torsion elastic modulus of the light chain domain. This work was supported by NIH R01AR032961 and R37AG26160 to DDT. YS was supported by NIH T32AG29796 and a University of Minnesota Interdisciplinary Doctoral Fellowship.