Backbone Orientation and Distance Measurements in Myosin II: Applications of High-Resolution EPR using a Bifunctional Spin Label

Abstract

We have used electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to obtain high-resolution measurements of individual structural elements within the myosin II catalytic domain (CD). Two complementary EPR techniques were employed to measure protein orientation (conventional EPR) and intra-protein distances (dipolar electron-electron resonance, DEER). The use of BSL greatly enhances the resolution of EPR, by virtue of its strongly immobilized and stereoselective 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. 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 ADP binding was clearly detected by EPR, and subsequently modeled using the orientation and distance measurements as constraints. We find that the structural change induced by ADP in the actin-bound myosin CD is clearly different from that predicted from actin-free crystal structures. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).