Structural Transitions of Myosin Associated with Force Generation in Spin-labeled Muscle Fibers

Muscle contraction is driven by the actin-activated hydrolysis of ATP by myosin, resulting in the relative sliding of actin and myosin filaments. Current models propose that filament sliding is driven by a structural transition of myosin's catalytic domain (CD) and light chain domain (LCD). The goal of this research is to measure structural transitions of myosin II (muscle and nonmuscle) that are associated for force generation. Structural measurements were made using electron paramagnetic resonance (EPR) spectroscopy. This work is comprised of two separate, but related, projects.;In the first project (Chapter 3), thiol crosslinking and EPR were used to resolve structural transitions of myosin's LCD and CD that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain (RLC) for endogenous RLC, with full retention of function. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), post-hydrolysis intermediate (A.M'.D.P), and rigor (A.M.D). To trap myosin in a structural state analogous to the elusive post-hydrolysis ternary complex A.M'.D.P, we used pPDM to crosslink SH1 (Cys707) to SH2 (Cys697) on the CD. EPR showed that the LCD of crosslinked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to crosslink SH1 to SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 crosslinking traps a state in which both the LCD and CD are in a structural state intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the crosslinked state is an A.M'.D.P analog on the force generation pathway.;In the second project, we present a method for obtaining high-resolution structural information of proteins using EPR of a bifunctional spin label (BSL). Two complimentary EPR techniques were employed to measure dynamics and orientation (conventional EPR) and intraprotein distances (dipolar electron-electron resonance). The exploitation of BSL is a key feature of this work. BSL attaches at residue positions i and i+4, which drastically restricts probe motion compared to monofunctional probes. For comparison, measurements were also made with the monofunctional spin label MSL. Subfragment 1 of Dictyostelium myosin II (S1dC) was used to exemplify the increased resolution provided by BSL. Using this approach, we demonstrate with experiments that BSL significantly increases resolution when measuring distance and orientation compared to MSL. And while this work does focus on the methodology, there is significant biological insight into myosin's nucleotide-dependent structural transitions.