Characterizing Protein/ligand Binding by DsC

All proteins are capable of recognizing and binding specific molecules such as other proteins, cofactors, prosthetic groups or drugs. Efforts to understand the mechanisms controlling selective binding were initially prompted by the realization that recognition and binding are universal features of all biochemical processes. These efforts have intensified with the awareness that knowledge-based drug design requires not only high-quality structural data on both the protein and the drug candidate, but also a quantitative understanding of the thermodynamics driving binding. A ligand will bind to a protein (or other macromolecule) only if the resulting complex is more stable than the original, non-liganded protein. Binding can occur to the native, folded protein (stabilizing the native state), or it can bind preferentially to the denatured protein, in which case the ligand will destabilize the native protein. In either case, binding triggers changes in intramolecular and intermolecular interactions, and in the dynamics of both the protein and the ligand. Since the degree of stabilization or destabilization of the native protein depends on the magnitude of the binding energy, comparison of the stability of the complex with the stability of the ligand-free protein allows the binding energy to be estimated. Differential scanning calorimetry (DSC) is particularly suited to studying the thermodynamics controlling conformational transitions in macromolecules such as proteins. As explained in the DSC overview note, DSC is generally used to measure the partial molar heat capacity of a protein over a temperature range of approximately 80 oC. If a ligand binds preferentially to the native state of the protein, the temperature at which the protein-ligand complex denatures will be higher compared to the temperature at which the free protein unfolds. DSC thus provides a direct measure of whether ligand binding to a protein is stabilizing or destabilizing, and so can complement studies of binding equilibria obtained by isothermal titration calorimetry (ITC).