title: Extraction of Thermodynamic Parameters of Protein Unfolding Using Parallelized Differential Scanning Fluorimetry.
abstract: Thermodynamic properties of protein unfolding have been extensively studied; however, the methods used have typically required significant preparation time and high protein concentrations. Here we present a facile, simple, and parallelized differential scanning fluorimetry (DSF) method that enables thermodynamic parameters of protein unfolding to be extracted. This method assumes a two-state, reversible protein unfolding mechanism and provides the capacity to quickly analyze the biophysical mechanisms of changes in protein stability and to more thoroughly characterize the effect of mutations, additives, inhibitors, or pH. We show the utility of the DSF method by analyzing the thermal denaturation of lysozyme, carbonic anhydrase, chymotrypsin, horseradish peroxidase, and cellulase enzymes. Compared with similar biophysical analyses by circular dichroism, DSF allows for determination of thermodynamic parameters of unfolding while providing greater than 24-fold reduction in experimental time. This study opens the door to rapid characterization of protein stability on low concentration protein samples.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Thaiesha A
authors.initials: TA
authors.lastname: Wright
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Jamie M
authors.initials: JM
authors.lastname: Stewart
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Richard C
authors.identifier: http://orcid.org/0000-0002-3006-3171
authors.initials: RC
authors.lastname: Page
authors.affiliation: Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States.
authors.forename: Dominik
authors.identifier: http://orcid.org/0000-0002-3828-5481
authors.initials: D
authors.lastname: Konkolewicz