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Single-Molecule Force-Clamp Spectroscopy: Dwell Time Analysis and Practical Considerations

TitleSingle-Molecule Force-Clamp Spectroscopy: Dwell Time Analysis and Practical Considerations
Publication TypeJournal Article
Year of Publication2011
AuthorsCao, Y, Li, H
Date PublishedFEB 15

Single-molecule force-clamp spectroscopy has become a powerful tool for studying protein folding/unfolding, bond rupture, and enzymatic reactions. Different methods have been developed to analyze force-clamp spectroscopy data on polyproteins to obtain kinetic parameters characterizing the mechanical unfolding of proteins, which are often modeled as a two-state process (a Poisson process). However, because of the finite number of domains in polyproteins, the statistical analysis, of the force-clamp spectroscopy data is different from that of a classical Poisson process, and the equivalency of different analysis methods remains to be proven. In this article, we show that these methods are equivalent and lead to accurate measurements of the unfolding rate constant. We also demonstrate that distinct from the constant-pulling-velocity experiments, in which the unfolding rate extracted from the data is dependent on the number of protein domains in the polyproteins (the N effect), force-clamp experiments do not show any N effect. Using a simulated data set, we also highlighted important practical considerations that one needs to take into account when using the single-molecule force-clamp spectroscopy technique to characterize the unfolding energy landscape of proteins.