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Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding

1
Department of Chemistry and Biochemistry, DePauw University, Greencastle, IN 46135, USA, [email protected]
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Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA 94720, USA
3
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
4
Department of Chemistry, University of California, Berkeley, CA 94720, USA
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Chan Zuckerburg Biohub, San Francisco, CA 94158, USA
*
Author to whom correspondence should be addressed.
Methods Protoc. 2019, 2(2), 32; https://doi.org/10.3390/mps2020032
Received: 11 March 2019 / Revised: 10 April 2019 / Accepted: 15 April 2019 / Published: 20 April 2019
(This article belongs to the Special Issue Single-Molecule Techniques)
While single-molecule force spectroscopy has greatly advanced the study of protein folding, there are limitations to what can be learned from studying the effect of force alone. We developed a novel technique, chemo-mechanical unfolding, that combines multiple perturbants—force and chemical denaturant—to more fully characterize the folding process by simultaneously probing multiple structural parameters—the change in end-to-end distance, and solvent accessible surface area. Here, we describe the theoretical background, experimental design, and data analysis for chemo-mechanical unfolding experiments probing protein folding thermodynamics and kinetics. This technique has been applied to characterize parallel protein folding pathways, the protein denatured state, protein folding on the ribosome, and protein folding intermediates. View Full-Text
Keywords: force spectroscopy; optical tweezers; chemo-mechanical unfolding; protein folding; denaturant; urea force spectroscopy; optical tweezers; chemo-mechanical unfolding; protein folding; denaturant; urea
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MDPI and ACS Style

Guinn, E.J.; Marqusee, S. Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding. Methods Protoc. 2019, 2, 32.

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