Energetics of ion-protein interactions

In keeping with the goals of our laboratory, efforts in this thesis are directed towards improving our understanding, and therefore our ability to calculate, the energetics of protein-ligand interactions. Electrostatic contributions to protein-ligand binding events are poorly understood, and underrepresented in data sets used to parameterize the energetics of protein unfolding and binding. Therefore, the focus in this thesis is placed on ion-protein interactions as model systems that can give insight into the contribution of charge-charge interactions to the enthalpy, entropy, and heat capacity changes associated with binding. In order to measure the energetics of charge-charge interactions, both differential scanning calorimetry and isothermal titration calorimetry are employed.

The use of linked equilibria to determine binding energetics for both extremely tight, and extremely weak binding events is described in the context of ligand binding linked to protein unfolding. The implications for drug screening methods based on protein unfolding are discussed. The theoretical development is then used to measure ion binding to proteins in two different systems that exhibit very different ion binding sites and system features.

The first system involves anion binding to a protein-protein complex, in which the binding site is formed when the protein-protein complex is formed. Binding of phosphate and sulfate occur with the same energetics, indicating that net charge is not dominating the observed energetics. Further, no salt-dependence to the binding of anions is observed. In the second system ions bind to the active site of a ribonuclease. Again, phosphate and sulfate bind to the ribonuclease with the same energetics, however comparing the energetics of binding for these anions between systems reveals differences in the energetic profiles. Further, in the ribonuclease case, there is a strong salt-dependence observed for the binding of a nucleotide inhibitor. The apparent discrepancies in the observed energetics and salt-dependencies in these systems can be resolved by considering the role of desolvation upon binding as well as the binding site geometries. This analysis leads to important considerations for interpreting an observed salt-dependence to a binding event. Furthermore, it is indicated that the current structure-based energetics calculations underestimate the contributions arising from charge-charge interactions.