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Force-Dependent Stepping Kinetics of Myosin-V

Anabel E.-M. Clemen ^*, Mojca Vilfan , Johann Jaud ^*, Junshan Zhang ^*, Michael Bärmann ^* and Matthias Rief ^*

^* Physics Department E22, Technical University Munich, 85747 Garching, Germany; and J. Stefan Institute, 1000 Ljubljana, Slovenia

Correspondence: Address reprint requests to Matthias Rief, E-mail: mrief{at}ph.tum.de.

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.

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