High-speed optical tweezers for the study of single molecular motors

Mechanical transitions in molecular motors often occur on a sub-millisecond time scale and rapidly follow binding of the motor with its cytoskeletal filament. Interactions of non-processive 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. Ultra-fast force-clamp spectroscopy is a constant-force optical tweezers technique that allows probing such rapid mechanical transitions and sub-millisecond kinetics of bio-molecular interactions, which can be particularly valuable for the study of non-processive motors, single heads of processive motors, or stepping dynamics of processive motors. Here we describe a step-by-step protocol for the application of ultra-fast 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 sub-nm spatial resolution. The protocol can be easily generalized to other families of motor proteins.