Protein Unfolding Coupled to Ligand Binding: Differential Scanning Calorimetry Simulation Approach

The aim of this work is to present the physicochemical basis underlying the changes in protein thermostability upon ligand binding. The article is addressed to advanced undergraduate and postgraduate chemistry students with an interest in protein biophysics. In addition, this article provides a useful tool for both learning and teaching biophysics because it links fundamental concepts: thermodynamics, chemical equilibrium, and protein stability. The influence of protein–ligand interactions on thermally-induced protein denaturation was monitored by differential scanning calorimetry (DSC). The changes in DSC output (thermogram) emerge by linking binding equilibrium with reversible protein unfolding thermodynamics. We derive the formalism for the description of protein unfolding in the presence of ligand that can bind to a single site on either native, unfolded, or both protein states. In addition to a rigorous mathematical description of the involved equilibria, the model provides the general formulation for simulating thermograms and calculating the changes in protein species during heating. First, we describe ligand interaction and emphasize the relationship between protein stability parameters and redistribution of species in equilibrium. After that, we describe the origin of bimodal thermograms, and finally, the effect on thermogram shape of protein concentration at constant ligand/protein mole ratio.