Structural Transitions in Myosin I Detected by Conventional and Pulsed EPR of a Bifunctional Spin Label

Abstract

We have employed electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to develop high-resolution constraints for the myosin catalytic domain in the presence and absence of actin. Two complementary EPR techniques were employed to measure protein orientation (continuous-wave EPR, CW-EPR) and intra-protein distances (double electron-electron resonance, DEER). The use of BSL greatly enhances the resolution of both techniques, by virtue of its strongly immobilized and stereospecific bifunctional attachment to the protein backbone at two engineered Cys residues. Crucially, both techniques utilized here permit the elucidation of myosin structure while in complex with actin, generating relevant constraints for the refinement of actomyosin structural models, and providing insight for structural changes induced by formation of the complex. In the current work, Dictyostelium myosin II was used as our model system. We measured nucleotide-dependent structural transitions of three key helices within the myosin CD. Three double-Cys sites were engineered, with Cys pairs located on the relay helix, helix HK (upper 50kDa domain) and helix HW (lower 50kDa domain), respectively. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin; BSL on a construct with two pairs was used to measure interprobe distances. The effect of MgADP binding was clearly detected by EPR, and subsequently modeled using the orientation and distance measurements as constraints. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).