Biochemical and Biophysical Research Communications
The thermal shift assay (TSA) is a powerful tool used to detect molecular interactions between proteins and ligands. Using temperature as a physical denaturant and an extrinsic fluorescent dye, the TSA tracks protein unfolding. This method precisely determines the midpoint of the unfolding transition (Tm" role="presentation">), which can shift upon the addition of a ligand. Though experimental protocols have been well developed, the thermal shift assay data traditionally yielded qualitative results. Quantitative methods for Kd" role="presentation"> determination relied either on empirical and inaccurate usage of Tm" role="presentation"> or on isothermal approaches, which do not take full advantage of the melting point precision provided by the TSA. We present a new analysis method based on a model that relies on the equilibrium system between the native and molten globule state of the protein using the van't Hoff equation. We propose the Kd" role="presentation"> can be determined by plotting Tm" role="presentation"> values versus the logarithm of ligand concentrations and fitting the data to an equation we derived. After testing this procedure with the monomeric maltose-binding protein and an allosterically regulated homotetrameric enzyme (ADP-glucose pyrophosphorylase), we observed that binding results correlated very well with previously established parameters. We demonstrate how this method could potentially offer a broad applicability to a wide range of protein classes and the ability to detect both active and allosteric site binding compounds.
Bhayani, Jaina A. and Ballicora, Miguel. Determination of dissociation constants of protein ligands by thermal shift assay. Biochemical and Biophysical Research Communications, 590, : 1-6, 2022. Retrieved from Loyola eCommons, Chemistry: Faculty Publications and Other Works, http://dx.doi.org/10.1016/j.bbrc.2021.12.041
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
© 2021 The Authors.