The effect of denaturants on protein thermal stability analyzed through a theoretical model considering multiple binding sites.

A novel mathematical development applied to protein ligand binding thermodynamics is proposed, which allows the simulation, and therefore the analysis of the effects of multiple and independent binding sites to the Native and/or Unfolded protein conformations, with different binding constant values. Protein stability is affected when it binds to a small number of high affinity ligands or to a high number of low affinity ligands. Differential scanning calorimetry (DSC) measures released or absorbed energy of thermally induced structural transitions of biomolecules. This paper presents the general theoretical development for the analysis of thermograms of proteins obtained for n-ligands bound to the native protein and m-ligands bound to their unfolded form. In particular, the effect of ligands with low affinity and with a high number of binding sites (n and/or m > 50) is analyzed. If the interaction with the native form of the protein is the one that predominates, they are considered stabilizers and if the binding with the unfolded species predominates, it is expected a destabilizing effect. The formalism presented here can be adapted to fitting routines in order to simultaneously obtain the unfolding energy and ligand binding energy of the protein. The effect of guanidinium chloride on bovine serum albumin thermal stability, was successfully analyzed with the model considering low number of middle affinity binding sites to the native state and a high number of weak binding sites to the unfolded state.