LAUSR

We have performed electron paramagnetic resonance (EPR) on actin-bound myosin labeled with a bifunctional spin label (BSL), in oriented muscle fibers. Our goal was to determine the effect of known small-molecule effectors on the structure of the complex of the myosin II catalytic domain (CD) bound to actin. The use of BSL in our EPR experiments greatly enhances the resolution of orientation and interspin distance measurements, due to the stereospecific bifunctional attachment to the protein backbone at two engineered Cys residues. We used Dictyostelium myosin II with Cys labeling sites as a model. Three pairs of Cys residues were engineered on our construct, which were located on relay helix, K-helix (upper 50 kDa domain), and W-helix (lower 50 kDa domain). Skinned muscle fiber bundles (actin filaments), decorated with spin-labeled myosin in the absence of nucleotide (“rigor”), were oriented parallel to the spectrometer's applied magnetic field. This procedure enables measurement of the orientation of BSL with respect to actin, which in turn gives us the orientation of individual myosin II structural elements. We tested several known myosin effectors, including arachidonic acid (AA). We observed a significant change in the structure of actin-bound myosin induced by AA, consistent with a change in the actin-activated ATPase activity of the spin-labeled construct. Our goal is to understand the structural changes in the myosin II CD in the presence of activators and inhibitors of actomyosin. This work was supported by NIH grant AR32961 to DDT.