High-Speed Optical Tweezers for the Study of Single Molecular Motors.

Mechanical transitions in molecular motors often occur on a submillisecond time scale and rapidly follow binding of the motor with its cytoskeletal filament. Interactions of nonprocessive molecular motors with their filament can be brief and last for few milliseconds or fraction of milliseconds. The investigation of such rapid events and their load dependence requires specialized single-molecule tools. Ultrafast force-clamp spectroscopy is a constant-force optical tweezers technique that allows probing such rapid mechanical transitions and submillisecond kinetics of biomolecular interactions, which can be particularly valuable for the study of nonprocessive motors, single heads of processive motors, or stepping dynamics of processive motors. Here we describe a step-by-step protocol for the application of ultrafast force-clamp spectroscopy to myosin motors. We give indications on optimizing the optical tweezers setup, biological constructs, and data analysis to reach a temporal resolution of few tens of microseconds combined with subnanometer spatial resolution. The protocol can be easily generalized to other families of motor proteins.