Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke

Abstract

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 μs, allows detection of interactions as brief as ∼100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2–1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.

DOI: 10.1038/nmeth.2152

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@article{Capitanio2012UltrafastFS, title={Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke}, author={Marco Capitanio and Monica Canepari and Manuela Maffei and Diego Beneventi and Carina Monico and Francesco Vanzi and Roberto Bottinelli and Francesco Pavone}, journal={Nature Methods}, year={2012}, volume={9}, pages={1013-1019} }