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Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy

Nature Protocols volume 8, pages 1261–1276 (2013)Cite this article

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Abstract

Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. A calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. The use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. These experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. These data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.

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Figure 1: Force-clamp AFM.
Figure 2: Calibration of AFM cantilevers.
Figure 3: Covalent attachment chemistry via HaloTag protein for AFM measurements.
Figure 4: Cropping force-clamp traces.
Figure 5: A walkthrough from cut traces to measured rates.
Figure 6: Illustration of possible problems during an AFM force-clamp experiment.
Figure 7: Summary of possible artifacts in force-clamp AFM.
Figure 8: Expected results using force-clamp AFM.

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Acknowledgements

We acknowledge all the past and present members of the Fernandez laboratory for their contribution in developing the AFM force-clamp technique. We acknowledge Luigs & Neumann for the pictures of the AFM setup and R.T. Sauer from Massachusetts Institute of Technology for the ERL-competent cells. This work was supported by grants from the US National Institutes of Health (HL066030 and HL061228 to J.M.F.). I.P. acknowledges the Swiss National Science Foundation for a postdoctoral research grant. J.A-.C. acknowledges a Fellowship from Fundación Ibercaja.

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Author notes

  1. Ionel Popa and Pallav Kosuri: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Columbia University, New York, USA

    Ionel Popa, Pallav Kosuri, Jorge Alegre-Cebollada & Julio M Fernandez

  2. Department of Physics, King's College London, London, UK

    Sergi Garcia-Manyes

  3. Randall Division of Cell and Molecular Biophysics, King's College London, London, UK

    Sergi Garcia-Manyes

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  1. Ionel Popa
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  2. Pallav Kosuri
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  3. Jorge Alegre-Cebollada
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  4. Sergi Garcia-Manyes
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  5. Julio M Fernandez
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Contributions

All the authors performed the measurements and wrote the manuscript.

Corresponding authors

Correspondence to Ionel Popa, Pallav Kosuri or Julio M Fernandez.

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Columbia University has licensed intellectual property to Luigs & Neumann GmbH.

Supplementary information

Supplementary Data

Raw data set for the mechanical unfolding of I27 in the presence of 30% glycerol, measured at four different forces. This is a single typical experiment that shows the full spectrum of challenges encountered when analyzing force-clamp measurements. (ZIP 278650 kb)

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Popa, I., Kosuri, P., Alegre-Cebollada, J. et al. Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy. Nat Protoc 8, 1261–1276 (2013). https://doi.org/10.1038/nprot.2013.056

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