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High-Speed Optical Tweezers for the Study of Single Molecular Motors

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Molecular Motors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1805))

Abstract

Mechanical transitions in molecular motors often occur on a submillisecond time scale and rapidly follow binding of the motor with its cytoskeletal filament. Interactions of nonprocessive molecular motors with their filament can be brief and last for few milliseconds or fraction of milliseconds. The investigation of such rapid events and their load dependence requires specialized single-molecule tools. Ultrafast force-clamp spectroscopy is a constant-force optical tweezers technique that allows probing such rapid mechanical transitions and submillisecond kinetics of biomolecular interactions, which can be particularly valuable for the study of nonprocessive motors, single heads of processive motors, or stepping dynamics of processive motors. Here we describe a step-by-step protocol for the application of ultrafast force-clamp spectroscopy to myosin motors. We give indications on optimizing the optical tweezers setup, biological constructs, and data analysis to reach a temporal resolution of few tens of microseconds combined with subnanometer spatial resolution. The protocol can be easily generalized to other families of motor proteins.

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Acknowledgments

We thank Dr. Claudia Arbore for assistance in the preparation of biological samples. The research leading to these results has received funding from the European Union Horizon 2020 Programme (H2020-INFRAIA-2014-2015 under grant agreement no. 654148—LASERLAB-EUROPE), by the Italian Ministry of University and Research (FIRB 2011 grant no. RBAP11×42L006, FIRB “Futuro in Ricerca” 2013 grant no. RBFR13V4M2, and Flagship Project NANOMAX), and by Ente Cassa di Risparmio di Firenze.

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Authors and Affiliations

  1. LENS—European Laboratory for Non-linear Spectroscopy, Sesto Fiorentino, Italy

    L. Gardini, A. Tempestini, F. S. Pavone & M. Capitanio

  2. National Institute of Optics—National Research Council, Florence, Italy

    L. Gardini & F. S. Pavone

  3. Department of Physics and Astronomy, University of Florence, Sesto Fiorentino, Italy

    A. Tempestini, F. S. Pavone & M. Capitanio

Authors
  1. L. Gardini
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  2. A. Tempestini
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  3. F. S. Pavone
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  4. M. Capitanio
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Corresponding author

Correspondence to M. Capitanio .

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Editors and Affiliations

  1. National Museum of Natural History, CNRS UMR7196 / INSERM U1154, Paris, France

    Christophe Lavelle

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Gardini, L., Tempestini, A., Pavone, F.S., Capitanio, M. (2018). High-Speed Optical Tweezers for the Study of Single Molecular Motors. In: Lavelle, C. (eds) Molecular Motors. Methods in Molecular Biology, vol 1805. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8556-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-8556-2_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8554-8

  • Online ISBN: 978-1-4939-8556-2

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