PKA Values of Buried Groups in Proteins are Sensitive to the Global Thermodynamic Stability

Abstract

Ionizable groups buried in hydrophobic environments in proteins are essential for all forms of biological energy transduction. The molecular determinants of the pKa values of these internal groups are poorly understood. It is increasingly apparent that conformational reorganization coupled to the ionization of the buried group is a major determinant of these pKa values. Specifically, the creation of charge in hydrophobic environments can trigger a shift from the fully folded state to local or partially unfolded states in which the charge can gain access to water or to an environment where the charge can be solvated. These alternative conformational states are not normally populated owing to the large free energy difference between the alternative and fully-folded native states; however, the partially unfolded states can become the new ground state under pH conditions where the internal group is charged. If the ionization of an internal group promotes the transition to a new conformational state then its pKa should be sensitive to the global thermodynamic stability (ΔG°) of the protein because this determines the energy gap between the ground and the alternative states. This was tested by measuring the pKa of two internal Lys residues in variants of staphylococcal nuclease with thermodynamic stabilities ranging from 8.4 to 13.8 kcal/mol. The magnitude of the shift in the pKa of the internal Lys residues was found to be sensitive to the ΔG° of the protein confirming that the pKa values of these Lys residues are determined by the probability of structural reorganization more than by local dielectric properties of their microenvironments. These observations imply that structure-based pKa calculations for buried groups and other electrostatic processes in hydrophobic environments require accurate treatment of conformational reorganization, which remains an extremely challenging proposition.