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Biochemical and Biophysical Research Communications





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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.


Author Posting © 2021 The Authors. This is an open access article distributed under the terms of the Creative Commons CC-BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The article was published in Biochemical and Biophysical Research Communications, Volume 590, January 2022,

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