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


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 ?-value analyses.

SUBMITTER: Wang Q 

PROVIDER: S-EPMC4706425 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Wang Qiang Q   Waterhouse Nicklas N   Feyijinmi Olusegun O   Dominguez Matthew J MJ   Martinez Lisa M LM   Sharp Zoey Z   Service Rachel R   Bothe Jameson R JR   Stollar Elliott J EJ  

PloS one 20160108 1


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 o  ...[more]

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