A buried lysine that titrates with a normal pKa: Role of conformational flexibility at the protein–water interface as a determinant of pKavalues

Previously we reported that Lys, Asp, and Glu residues at positions 66 and 92 in staphylococcal nuclease (SNase) titrate with pKa values shifted by up to 5 pKa units in the direction that promotes the neutral state. In contrast, the internal Lys‐38 in SNase titrates with a normal pKa. The crystal structure of the L38K variant shows that the side chain of Lys‐38 is buried. The ionizable moiety is ∼7 Å from solvent and ion paired with Glu‐122. This suggests that the pKa value of Lys‐38 is normal because the energetic penalty for dehydration is offset by a favorable Coulomb interaction. However, the pKa of Lys‐38 was also normal when Glu‐122 was replaced with Gln or with Ala. Continuum electrostatics calculations were unable to reproduce the pKa of Lys‐38 unless the protein was treated with an artificially high dielectric constant, consistent with structural reorganization being responsible for the normal pKa value of Lys‐38. This reorganization must be local because circular dichroism and NMR spectroscopy indicate that the L38K protein is native‐like under all conditions studied. In molecular dynamics simulations, the ion pair between Lys‐38 and Glu‐122 is unstable. The simulations show that a minor rearrangement of a loop is sufficient to allow penetration of water to the amino moiety of Lys‐38. This illustrates both the important roles of local flexibility and water penetration as determinants of pKa values of ionizable groups buried near the protein–water interface, and the challenges faced by structure‐based pKa calculations in reproducing these effects.