Abstract
Muscles contract through actin-myosin interactions modulated by the ATPase reaction. To determine how muscle shortening is generated by actin-myosin biochemistry and kinetics, we are developing an in vitro motility assay that allows us to simultaneously measure actin sliding velocities, V, and actin-myosin attachment times, Ton. To measure Ton during a motility assay, we monitor changes in actin filament dynamics near a myosin binding site using nanometer tracking of actin filaments labeled with single qdots. Actin-myosin binding damps actin dynamics, and Ton is estimated from the average duration of these binding events. With this approach, we observe that the duration of actin-myosin binding events decreases linearly with increasing ATP concentrations, resulting in an estimated ATP induced dissociation rate constant of ∼9 μM−1·s−1, consistent with previous kinetic measurements. This technique allows us to determine the mechanochemistry of a single myosin head functioning within the context of many myosin molecules interacting with a single actin filament.
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