Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke
Authors: Capitanio M., Canepari M., Maffei M., Beneventi D., Monico C., Vanzi F., Bottinelli R., Pavone FS.
Autors Affiliation: Univ Florence, European Lab Nonlinear Spect, Sesto Fiorentino, Italy; Univ Florence, Dept Phys & Astron, Sesto Fiorentino, Italy; Univ Pavia, Dept Mol Med, I-27100 Pavia, Italy; Univ Florence, Dept Evolutionary Biol, Florence, Italy; Sci Inst Pavia, Fdn Salvatore Maugeri, Pavia, Italy; CNR, Natl Opt Inst, Florence, Italy; Int Ctr Computat Neurophoton, Sesto Fiorentino, Italy
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 similar to 10 mu s, allows detection of interactions as brief as similar to 100 mu s and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNDNA-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. Journal/Review: NATURE METHODS
Volume: 9 (10) Pages from: 1013 to: 1019
More Information: Publisher: NATURE PUBLISHING GROUP, MACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND
DOI: 10.1038/NMETH.2152 KeyWords: OPTICAL TWEEZERS; TRANSCRIPTION INITIATION; KINETICS; DNA TRANSLOCATION; BINDINGDOI: 10.1038/NMETH.2152Citations: 76data from “WEB OF SCIENCE” (of Thomson Reuters) are update at: 2020-02-23References taken from IsiWeb of Knowledge: (subscribers only)Connecting to view paper tab on IsiWeb: Click hereConnecting to view citations from IsiWeb: Click here