Myosin V exhibits a high duty cycle and large unitary displacement

Myosin V is a double-headed unconventional myosin that has been implicated in organelle transport. To perform this role, myosin V may have a high duty cycle. To test this hypothesis and understand the properties of this molec ule at the molecular level, we used the laser trap and in vitro motility as say to characterize the mechanics of heavy meromyosin-like fragments of myo sin V (M5(HMM)) expressed in the Baculovirus system. The relationship betwe en actin filament velocity and the number of interacting M5(HMM) molecules indicates a duty cycle of greater than or equal to 50%. This high duty cycl e would allow actin filament translocation and thus organelle transport by a few M5HMM molecules. Single molecule displacement data showed predominant ly single step events of 20 nm and an occasional second step to 37 nm. The 20-nm unitary step represents the myosin V working stroke and is independen t of the mode of M5(HMM) attachment to the motility surface or light chain content. The large M5HMM working stroke is consistent with the myosin V nec k acting as a mechanical lever. The second step is characterized by an incr eased displacement variance, suggesting a model for how the two heads of my osin V function in processive motion.