Stabilization of proteins by ligand binding: application to drug screening and determination of unfolding energetics.

The observed stability of a protein is altered when ligands bind, which results in a shift in the melting temperature (T(m)). Binding to the native state in the absence of binding to the denatured state will necessarily lead to an increase in the T(m), while binding to the unfolded state in the absence of native state binding will decrease the T(m) relative to that of the protein in the absence of ligand. These effects are required by the thermodynamics of reversible folding. However, the relationship between binding affinity and the magnitude of the observed temperature shift is not a simple correlation (i.e., a larger shift in T(m) does not necessarily mean tighter binding) and is complicated by interaction with the denatured state. Using exact simulations, the range of behavior for the dependence of the observed T(m) shift on the energetics of ligand binding is investigated here. Specifically, differential scanning calorimetry (DSC) curves are simulated for protein unfolding in the presence of ligands binding to both the native and denatured states. The results have implications for drug screening and the determination of heat capacity changes for protein unfolding.