Local Backbone Flexibility as a Determinant of the Apparent pK_a Values of Buried Ionizable Groups in Proteins

Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction. These groups can have highly anomalous pK_a values that reflect the incompatibility between charges and dehydrated environments. A systematic study of pK_a values of buried ionizable groups in staphylococcal nuclease (SNase) suggests that these pK_a values are determined in part by conformational reorganization of the protein. Lys-66 is one of the most deeply buried residues in SNase. We show that its apparent pK_a of 5.7 reflects the average of the pK_a values of Lys-66 in different conformational states of the protein. In the fully folded state, Lys-66 is deeply buried in the hydrophobic core of SNase and must titrate with a pK_a of ≪5.7. In other states, the side chain of Lys-66 is fully solvent-exposed and has a normal pK_a of ≈10.4. We show that the pK_a of Lys-66 can be shifted from 5.7 toward a more normal value of 7.1 via the insertion of flanking Gly residues at positions 64 and 67 to promote an "open" conformation of SNase. Crystal structures and nuclear magnetic resonance spectroscopy show that in these Gly-containing variants Lys-66 can access bulk water as a consequence of overwinding of the C-terminal end of helix 1. These data illustrate that the apparent pK_a values of buried groups in proteins are governed in part by the difference in free energy between different conformational states of the protein and by differences in the pK_a values of the buried groups in the different conformations.