The thermodynamic linkage of proton binding and protein function

This thesis describes the development and use of new experimental tools and computational techniques to assess the role of interactions between charged groups on the stability and activity of proteins. This stability and activity are quantitatively indicated by changes in free energy as a result of protein folding and ligand binding, respectively. The techniques of capillary electrophoresis (CE) and charge ladders measure the linkage between the cooperativity of proton binding and protein function. Monte Carlo simulations together with Poisson Boltzmann theory of continuum electrostatics are used to model this thermodynamic linkage. The combination of experiments and modeling provides general design principles for the effective engineering of proteins via interactions between charged groups.; The results of the experimental and computational work are consistent. In both protein folding and binding, relying on a single protonation state may not capture all of the physics occurring throughout the processes. Experimental studies on α-lactalbumin show a strong change in protonation state as a result of denaturation. Computational studies on barnase and barstar also reveal a change in the protonation states of barnase and barstar upon binding; this change is modeled accurately by allowing the observed protonation states to be averages of many protonation states in equilibrium. Thus, the observed change in protonation state is in reality a change in the distribution of ensembles of protonation states.; This thesis is intended to give the reader an idea of the significance with which the extent of cooperativity of proton binding can affect the structure and activity of a protein molecule. Since electrostatic interactions are long range, modifying a single site can affect many other titratable groups in the protein resulting in a large collective energetic impact. The consequences of this thesis are a starting point for the scientist to regulate cooperativity of a protein so that by modifying a few key residues, one can engineer protein function and stability without significantly altering the surface potential on the protein or the protein chemical composition.