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

Year: 2012

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: We thank G. Belcastro for his help with Lac repressor experiments, M. Giuntini for quadrant detector photodiode electronics, and V. Lombardi and L. Gardini for discussion. This research was funded by the EU Seventh Framework Programme (FP7/2007-2013; grant agreements B0 211383, B0 228334 and B0 241526), by the Italian Ministry of University and Research (PRIN 2006 2006051323_003, FIRB 2011 RBAP11X42L006 and Flagship Project NANOMAX) and by Ente Cassa di Risparmio di Firenze to F.S.P. and by the EU Seventh Framework Programme (FP7/2007-2013; grant agreement 223576, Combating age-related muscle weakness (MYOAGE)) to R.B.
KeyWords: Optical Tweezers; Rna-polymerase; Muscle Myosin; Transcription Initiation; Dna Translocation; Kinetics; Binding; Motor; Contraction; Elongation
DOI: 10.1038/NMETH.2152

Citations: 112
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