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.
Highlights
The study of protein folding kinetics and stability is central to understanding protein structure, dynamics and energetics
Our ultimate goal is to measure a large number of mutants in a variety of buffer conditions, making the development of a fully automated protein folding kinetic assay highly desirable
The unfolding kinetic constant for the AbpSH3 domain was expected to decrease when the ArkA binding peptide is covalently attached by a flexible linker as observed in other SH3 domain-peptide hybrids [21]
Summary
The study of protein folding kinetics and stability is central to understanding protein structure, dynamics and energetics. Whereas global stability of a protein measures the proportion of a protein that is in its native state at equilibrium, folding rates provide information on protein’s kinetic stability. Kinetic stability is important to consider under physiological conditions of the cell where a protein in the unfolded state may irreversibly aggregate or become proteolytically digested and lose its function [1]. The measurement of equilibrium and kinetic constants of these proteins allows a φ-value to be calculated with solely folding kinetic data or solely unfolding kinetic data (Eq 1). 1 A ð1Þ where kWu T and kMu UT are the unfolding kinetic constants of the wild type and mutant respectively, calculated from kinetic experiments and DDGou is the difference (WT—mutant) in their free.
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