12 September 2013 Ultra-fast force-clamp laser trapping of single molecular motors and DNA binding proteins
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Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay of only ∼10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.
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Marco Capitanio, Marco Capitanio, Carina Monico, Carina Monico, Francesco Vanzi, Francesco Vanzi, Francesco Saverio Pavone, Francesco Saverio Pavone, "Ultra-fast force-clamp laser trapping of single molecular motors and DNA binding proteins", Proc. SPIE 8810, Optical Trapping and Optical Micromanipulation X, 88101E (12 September 2013); doi: 10.1117/12.2023592; https://doi.org/10.1117/12.2023592

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