Impact of protein and ligand impurities on ITC-derived protein–ligand thermodynamics

• Protein–ligand interactions were characterized by isothermal titration calorimetry.
• The impact of concentration errors and impurities was investigated.
• Errors in protein content did not influence thermodynamic parameters.
• Errors in ligand concentration and impurities had significant effects.
• Correcting data of impure ligands was a pitfall for accurate thermodynamics.

BackgroundThe thermodynamic characterization of protein–ligand interactions by isothermal titration calorimetry (ITC) is a powerful tool in drug design, giving valuable insight into the interaction driving forces. ITC is thought to require protein and ligand solutions of high quality, meaning both the absence of contaminants as well as accurately determined concentrations.MethodsLigands synthesized to deviating purity and protein of different pureness were titrated by ITC. Data curation was attempted also considering information from analytical techniques to correct stoichiometry.Results and conclusionsWe used trypsin and tRNA-guanine transglycosylase (TGT), together with high affinity ligands to investigate the effect of errors in protein concentration as well as the impact of ligand impurities on the apparent thermodynamics. We found that errors in protein concentration did not change the thermodynamic properties obtained significantly. However, most ligand impurities led to pronounced changes in binding enthalpy. If protein binding of the respective impurity is not expected, the actual ligand concentration was corrected for and the thus revised data compared to thermodynamic properties obtained with the respective pure ligand. Even in these cases, we observed differences in binding enthalpy of about 4 kJ ⋅ mol− 1, which is considered significant.General significanceOur results indicate that ligand purity is the critical parameter to monitor if accurate thermodynamic data of a protein–ligand complex are to be recorded. Furthermore, artificially changing fitting parameters to obtain a sound interaction stoichiometry in the presence of uncharacterized ligand impurities may lead to thermodynamic parameters significantly deviating from the accurate thermodynamic signature.

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