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Development and Application of a High Throughput Protein Unfolding Kinetic Assay.

By Qiang Wang, Nicklas Waterhouse, Olusegun Feyijinmi, Matthew J Dominguez, Lisa M Martinez, Zoey Sharp, Rachel Service, Jameson R Bothe and Elliott J Stollar

Abstract

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses

Topics: Medicine, R, Science, Q
Publisher: Public Library of Science (PLoS)
DOI identifier: 10.1371/journal.pone.0146232
OAI identifier: oai:doaj.org/article:9413f6c09b5740f897d2e5ea8418b5c4
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