title: Muscle and nonmuscle myosins probed by a spin label at equivalent sites in the force-generating domain.
abstract: We have engineered a mutant of Dictyostelium discoideum (Dicty) myosin II that contains the same fast-reacting "SH1" thiol as in muscle myosin, spin-labeled it, and performed electron paramagnetic resonance (EPR) to compare the structure of the force-generating region of the two myosins. Dicty myosin serves as a model system for muscle myosin because of greater ease of mutagenesis, expression, and crystallization. The catalytic domains of these myosins have nearly identical crystal structures in the apo state, but there are significant differences in ATPase kinetics, and there are no crystal structures of skeletal muscle myosin with bound nucleotides, so another structural technique is needed. Previous EPR studies, with a spin label attached to SH1 in muscle myosin, have resolved the key structural states of this region. Therefore, we have performed identical experiments on both myosins spin-labeled at equivalent sites. Spectra were identical for the two myosins in the apo and ADP-bound states. With bound ADP and phosphate analogs, (i) both proteins exhibit two resolved structural states (prepowerstroke, postpowerstroke) in a single biochemical state (defined by the bound nucleotide), and (ii) these structural states are essentially identical in the two myosins but (iii) are occupied to different extents as a function of the biochemical state. We conclude that (i) myosin structural and biochemical states do not have a one-to-one correspondence, and (ii) Dicty myosin can serve as a good analog for structural studies of muscle myosin only if differences in the coupling between biochemical and structural states are taken into account.
authors.affiliation: Departments of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
authors.forename: Roman V
authors.initials: RV
authors.lastname: Agafonov
authors.forename: Yuri E
authors.initials: YE
authors.lastname: Nesmelov
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Titus
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas