title: Global fit analysis of myosin-5b motility reveals thermodynamics of Mg2+-sensitive acto-myosin-ADP states.
abstract: Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our investigation of temperature and free Mg(2+)-ion dependencies of sliding velocities of a high duty ratio class-5 myosin motor, myosin-5b from D. discoideum using in vitro motility assays. Previous studies have shown that the sliding velocity of class-5 myosins obeys modulation by free Mg(2+)-ions. Free Mg(2+)-ions affect ADP release kinetics and the dwell time of actin-attached states. The latter determines the maximal velocity of actin translocation in the sliding filament assay. We measured the temperature dependence of sliding velocity in the range from 5 to 55°C at two limiting free Mg(2+)-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg(2+)-ions and non-linearity of the temperature dependence of sliding velocity. According to this model, velocity is represented as a simple analytical function of temperature and free Mg(2+)-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20°C) dependence of sliding velocity. As a result we obtain thermodynamic parameters (ΔH(Mg) and ΔS(Mg)) of a fast equilibrium between magnesium free (AM·D) and magnesium bound acto-myosin-ADP (AM· Mg(2+)D) states and the corresponding enthalpic barriers associated with ADP release (ΔH1(‡) and ΔH2(‡)). The herein presented integrative approach of data analysis based on global fitting can be applied to the remaining steps of the acto-myosin ATPase cycle facilitating the determination of energetic parameters and thermodynamics of acto-myosin interactions.
authors.affiliation: Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany.
authors.forename: Igor
authors.initials: I
authors.lastname: Chizhov
authors.forename: Falk K
authors.initials: FK
authors.lastname: Hartmann
authors.forename: Nikolas
authors.initials: N
authors.lastname: Hundt
authors.forename: Georgios
authors.initials: G
authors.lastname: Tsiavaliaris
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D004023:Dictyostelium / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D024701:Myosin Type V / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D000244:Adenosine Diphosphate; D024701:Myosin Type V; D008274:Magnesium