%0 Journal Article
%T Global Fit Analysis of Myosin-5b Motility Reveals Thermodynamics of Mg2+-Sensitive Acto-Myosin-ADP States
%A Igor Chizhov
%A Falk K. Hartmann
%A Nikolas Hundt
%A Georgios Tsiavaliaris
%J PLOS ONE
%D 2013
%I Public Library of Science (PLoS)
%R 10.1371/journal.pone.0064797
%X 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 Mg2+-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 Mg2+-ions. Free Mg2+-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¡ãC at two limiting free Mg2+-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg2+-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 Mg2+-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20¡ãC) dependence of sliding velocity. As a result we obtain thermodynamic parameters (¦¤HMg and ¦¤SMg) of a fast equilibrium between magnesium free (AM¡¤D) and magnesium bound acto-myosin-ADP (AM¡¤ Mg2+D) states and the corresponding enthalpic barriers associated with ADP release (¦¤H1£¿ and ¦¤H2£¿). 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.
%U http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0064797