A coupled ionization-conformational equilibrium is required to understand the properties of ionizable residues in the hydrophobic interior of staphylococcal nuclease

Ionizable residues in the interior of proteins play essential roles, especially in biological energy transduction, but are relatively rare and seem incompatible with the complex and polar environment. We perform a comprehensive study of the internal ionizable residues on 21 variants of staphylococcal nuclease with internal Lys, Glu, or Asp residues. Using pH replica exchange molecular dynamics simulations, we find that, in most cases, the pK_a values of these internal ionizable residues are shifted significantly from their values in solution. Our calculated results are in excellent agreement with the experimental observations of the Garcia-Moreno group. We show that the interpretation of the experimental pK_a values requires the study of not only protonation changes but also conformational changes. The coupling between the protonation and conformational equilibria suggests a mechanism for efficient pH-sensing and regulation in proteins. This study provides new physical insights into how internal ionizable residues behave in the hydrophobic interior of proteins.