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

Nature Methods volume 9, pages 10131019 (2012) | Download Citation

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.

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Acknowledgements

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.

Author information

Affiliations

  1. European Laboratory for Non-linear Spectroscopy, University of Florence, Sesto Fiorentino, Italy.

    • Marco Capitanio
    • , Diego Beneventi
    • , Carina Monico
    • , Francesco Vanzi
    •  & Francesco Saverio Pavone
  2. Department of Physics and Astronomy, University of Florence, Sesto Fiorentino, Italy.

    • Marco Capitanio
    •  & Francesco Saverio Pavone
  3. Department of Molecular Medicine, University of Pavia, Pavia, Italy.

    • Monica Canepari
    • , Manuela Maffei
    •  & Roberto Bottinelli
  4. Department of Evolutionary Biology, University of Florence, Florence, Italy.

    • Francesco Vanzi
  5. Fondazione Salvatore Maugeri, Scientific Institute of Pavia, Pavia, Italy.

    • Roberto Bottinelli
  6. National Institute of Optics–National Research Council, Florence, Italy.

    • Francesco Saverio Pavone
  7. International Center of Computational Neurophotonics, Sesto Fiorentino, Italy.

    • Francesco Saverio Pavone

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Contributions

M. Capitanio conceived and designed the ultrafast force clamp, performed experiments, analyzed data and wrote the paper. M.M. prepared samples and contributed to myosin experiments. D.B. contributed to setting up the ultrafast force clamp and performing myosin experiments. C.M. prepared samples and performed LacI experiments. F.V. supervised LacI experiments and wrote the paper. R.B. and M. Canepari supervised myosin experiments. R.B. contributed to writing the paper. F.S.P. supervised the design of the ultrafast force-clamp experiments and the whole project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Marco Capitanio.

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    Supplementary Figures 1–4, Supplementary Notes 1–8, Supplementary Results

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DOI

https://doi.org/10.1038/nmeth.2152

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