Determination of Protein–Ligand Binding Affinities by Thermal Shift Assay

Quantification of protein-ligand interactions is crucial for understanding the protein's biological function and for drug discovery. In this study, we employed three distinct approaches for determination of protein-ligand binding affinities by a thermal shift assay using a single ligand concentration. We present the results of the comparison of the performance of the conventional curve fitting (CF) method and two newly introduced methods - assuming zero heat capacity change across small temperature ranges (ZHC) and utilizing the unfolding equilibrium constant (UEC); the latter has the advantage of reducing calculations by obtaining the unfolding equilibrium constant directly from the experimental data. Our results highlight superior performance of the ZHC and UEC methods over the conventional CF method in estimating the binding affinity, irrespective of the ligand concentration. In addition, we evaluated how the new methods can be applied to high-throughput screening for potential binders, when the enthalpy (Δ<i>H</i> _L) and molar heat capacity change (Δ<i>C</i> _PL) of ligand binding are unknown. Our results suggest that, in this scenario, using the -300 cal K^-1 mol^-1 assumption for Δ<i>C</i> _pL and either -5 kcal mol^-1 or the average enthalpy efficiency-based estimation for Δ<i>H</i> _L(<i>T</i>) can still provide reasonable estimates of the binding affinity. Incorporating the new methods into the workflow for screening of small drug-like molecules, typically conducted using single-concentration libraries, could greatly simplify and streamline the drug discovery process.