Journal
NATURE METHODS
Volume 9, Issue 10, Pages 1013-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.2152
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Funding
- EU Seventh Framework Programme [B0 211383, B0 228334, B0 241526, 223576]
- Italian Ministry of University and Research [PRIN 2006 2006051323_003, FIRB 2011 RBAP11X42L006]
- Ente Cassa di Risparmio di Firenze
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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.
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