Development and Application of a High Throughput Protein Unfolding Kinetic Assay

Abstract

Protein folding and unfolding kinetics contribute to stability and provide important insights into protein structure and function. Traditionally, protein folding kinetics have been studied using stopped flow kinetics, a technique that is time intensive and specialized. Here, we present a simple high throughput protein folding kinetic assay in a plate reader that should be amenable to 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 assay with a plate reader equilibrium assay, to obtain indirect folding rates that also agree and use these approaches to characterize a AbpSH3-peptide hybrid. Our high throughput protein folding kinetic assays will save substantial amounts of time, allow more accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and will provide a means for future in-depth phi-value analyses.