Electrostatic Coupling and Conformational Fluctuations as Determinants of pKa Values in Proteins

Electrostatic effects, particularly proton binding and transfer, govern many essential biological functions of proteins. Relating protein structure to function therefore requires understanding the molecular determinants of pKa values in proteins. Various factors influence these pKa values, including hydration, hydrogen bonding, and Coulomb interactions. Resolving the contributions of these factors requires structure-based calculations of electrostatic energies. To be useful, such calculations must be able to reproduce experimental data, which current structure-based pKa calculations are unable to do. This work examined two problems where experimental insight was necessary to improve structure-based electrostatics calculations. Enzyme active sites typically contain clusters of ionizable residues, leading to strong electrostatic interactions and complex coupling between the pKa values of the residues involved. To better characterize these interactions, the ionizable residues clustered in the active site of staphylococcal nuclease (SNase) were systematically neutralized by mutagenesis, and the effect on the pKa values of the other ionizable groups was measured using NMR spectroscopy. One of the residues in the active site, Asp-19, has a depressed intrinsic pKa due to accepting a hydrogen bond, and is therefore insensitive to repulsive Coulomb interactions. Meanwhile, Asp-21 has an elevated intrinsic pKa due to acting as a hydrogen bond donor and therefore absorbs most of the repulsive interaction energy in the cluster. Therefore in systems with strong coupling between ionizable groups, small structural variations can lead to large differences in pKa values. Crystal structures may not be sufficiently accurate to capture these variations. It is believed that one reason for the failure of structure-based pKa calculations is that they do not explicitly include the effects of backbone reorganization. To show that backbone reorganization has a significant effect on pKa values, the pKa values of carboxylic groups in SNase were measured in the presence of glycine substitutions that perturbed the local stability of the protein backbone. Significant changes in pKa values were observed that could not be reproduced with calculations that treat the protein backbone as static. This suggests that structure-based electrostatics calculations need to account for backbone reorganization explicitly