期刊
PLOS ONE
卷 8, 期 5, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0064797
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资金
- Deutsche Forschungsgemeinschaft [TS 169/3-1, TS 169/3-2]
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 degrees C 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 degrees C) dependence of sliding velocity. As a result we obtain thermodynamic parameters (Delta H-Mg and Delta S-Mg) of a fast equilibrium between magnesium free (AM center dot D) and magnesium bound acto-myosin-ADP (AM center dot Mg2+D) states and the corresponding enthalpic barriers associated with ADP release (Delta H-1(double dagger) and Delta H-2(double dagger)). 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 actomyosin interactions.
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