Extraction of Protein Thermodynamic Parameters from High‐Throughput Differential Scanning Fluorimetry Assays

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 high‐throughput differential scanning fluorimetry (DSF) method that enables a complete set of 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 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. Compared to similar biophysical analyses by circular dichroism, DSF provides greater than 24‐fold reduction in experimental time. This study opens to door to rapid characterization of protein stability on low concentration protein samples.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP) and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).