title: Conformationally trapping the actin-binding cleft of myosin with a bifunctional spin label.
abstract: We have trapped the catalytic domain of Dictyostelium (Dicty) myosin II in a weak actin-binding conformation by chemically crosslinking two engineered cysteines across the actin-binding cleft, using a bifunctional spin label (BSL). By connecting the lower and upper 50 kDa domains of myosin, the crosslink restricts the conformation of the actin-binding cleft. Crosslinking has no effect on the basal ATPase activity of isolated myosin, but it impairs rigor actin binding and actin-activation of myosin ATPase. EPR spectra of BSL provide insight into actomyosin structural dynamics. BSL is highly immobilized within the actin-binding cleft and is thus exquisitely sensitive to the global orientation and rotational motions of the myosin head. Conventional EPR shows that myosin heads bound to oriented actin filaments are highly disordered with respect to the actin filament axis, in contrast to the nearly crystalline order of myosin heads in rigor. This disorder is similar to that of weakly bound heads induced by ATP, but saturation transfer EPR shows that the disorder of crosslinked myosin is at least 100 times slower. Thus this cleft-crosslinked myosin is remarkably similar, in both actin affinity and rotational dynamics, to SH1-SH2 crosslinked BSL-myosin S1. We conclude that, whether myosin is trapped at the actin-myosin interface or in the force-generating region between the active site and lever arm, the structural state of myosin is intermediate between the weak-binding state preceding phosphate release and the strong-binding state that succeeds it. We propose that it represents the threshold of force generation.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Moen
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Klein
mesh_terms: D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001696:Biomechanical Phenomena; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004023:Dictyostelium / Q000187:drug effects / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D008954:Models, Biological; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D011817:Rabbits; D012399:Rotation; D013113:Spin Labels
chemical_list: D000199:Actins; D003432:Cross-Linking Reagents; D013113:Spin Labels; D000205:Actomyosin; D009218:Myosins