PLoS ONE (Jan 2016)

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

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

DOI
https://doi.org/10.1371/journal.pone.0146232
Journal volume & issue
Vol. 11, no. 1
p. e0146232

Abstract

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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.