title: 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 ϕ-value analyses.
authors.affiliation: Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
authors.forename: Nicklas
authors.initials: N
authors.lastname: Waterhouse
authors.affiliation: Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
authors.forename: Olusegun
authors.initials: O
authors.lastname: Feyijinmi
authors.affiliation: Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Dominguez
authors.affiliation: Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Martinez
authors.affiliation: Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
authors.forename: Zoey
authors.initials: Z
authors.lastname: Sharp
authors.affiliation: Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
authors.forename: Rachel
authors.initials: R
authors.lastname: Service
authors.affiliation: NMRFAM, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Jameson R
authors.initials: JR
authors.lastname: Bothe
authors.affiliation: Department of Physical Sciences, Eastern New Mexico University, Portales, New Mexico, United States of America.
authors.forename: Elliott J
authors.initials: EJ
authors.lastname: Stollar
mesh_terms: D002850:Chromatography, Gel; D019791:Guanidine / Q000737:chemistry; D057166:High-Throughput Screening Assays; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008958:Models, Molecular; D011489:Protein Denaturation; D055550:Protein Stability; D059748:Proteolysis; D053838:Scattering, Small Angle; D012996:Solutions; D013816:Thermodynamics; D014961:X-Ray Diffraction; D018909:src Homology Domains
chemical_list: D008840:Microfilament Proteins; D012996:Solutions; D019791:Guanidine