Determination of Ligand Binding Affinity and Specificity of Purified START Domains by Thermal Shift Assays Using Circular Dichroism

Methods in Molecular Biology
Danny LétourneauPierre Lavigne

Abstract

The use of direct calorimetric methods such as isothermal titration calorimetry for measuring the affinity and specificity of protein-ligand interactions requires large amounts of proteins and ligands. When material is scarce and/or in the absence of calorimeters, thermal Shift Assays (TSA) using Circular Dichroism (CD) or other spectroscopic methods offers an alternative and quantitative method for the determination of apparent or indirect thermodynamical parameters describing the affinity of ligands for proteins. Indeed, the binding constants of ligands (Kb) and other parameters such as the enthalpy and Gibbs free energy of binding may be estimated from the changes in the stability curves ΔGu(T) of a protein in the presence of a ligand. Here we describe the application of two different procedures proposed by Layton and Hellinga et al. (Biochemistry 49:10831-10841, 2010) to evaluate the apparent Kb of testosterone to the START (StAR-related lipid transfer domain) domains.

Citations

May 22, 2020·The Journal of Biological Chemistry·Jobst LiebauLena Mäler
May 11, 2021·Frontiers in Molecular Biosciences·Xiuyan YangShaoyong Lu

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