title: Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain.
abstract: We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Muretta
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Petersen
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001696:Biomechanical Phenomena; D020134:Catalytic Domain; D003199:Computer Systems; D018360:Crystallography, X-Ray; D004023:Dictyostelium / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D024462:Myosin Type II / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D017433:Protein Structure, Secondary; D011817:Rabbits; D013997:Time Factors
chemical_list: D000199:Actins; D010710:Phosphates; D024462:Myosin Type II