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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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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All the authors performed the measurements and wrote the manuscript.
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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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DOI: https://doi.org/10.1038/nprot.2013.056
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