The pKa values of buried ionizable amino acids can be determined by the thermodynamic stability of the protein

Ionizable groups in hydrophobic environments in proteins usually titrate with anomalous pKa values. The ionization of these buried residues is often coupled to transitions between conformational states. Here we test the hypothesis that because the thermodynamic stability of a protein ({Delta}G{degrees}H2O) determines the probability of conformational transitions, the apparent pKa values of buried residues can be governed by {Delta}G{degrees}H2O. Variants of staphylococcal nuclease (SNase) with either Lys-66 or Lys-92 buried in its hydrophobic interior were engineered along with surface mutations that alter {Delta}G{degrees}H2O without affecting the electrostatic properties of the internal microenvironments of the buried Lys residues. The measured pKa values of these Lys residues largely correlates with {Delta}G{degrees}H2O. NMR spectroscopy was used to demonstrate that the structural changes of the protein backbone coupled to the ionization of Lys-66 or Lys-92 are comparable regardless of the {Delta}G{degrees}H2O of the protein. NMR spectroscopy confirmed that global unfolding of the Lys-92 variant coincides with the apparent pKa of the Lys side chain. The data presented show that the anomalous pKa values measured for internal residues in proteins do not necessarily report on local dielectric or electrostatic properties of the microenvironments around the ionizable group; rather, they can report on the energetics of pH-driven conformational transitions. These data suggest that accurate structure-based calculation of pKa values will require de novo prediction of partially unfolded conformations, and accurate calculation of free energy differences between conformational states, both of which remain formidable challenges.