PH-Driven Conformational Reorganization of Proteins: NMR Spectroscopy Study with Buried Lys Residues

Abstract

The pH sensitivity of proteins is essential for biological energy transduction and for cellular and physiological homeostasis. The thermodynamic basis of pH effects in proteins is well understood but detailed mechanistic understanding of how changes in pH drive conformational reorganization in proteins is not. This was addressed systematically using NMR spectroscopy to characterize pH-dependent conformational changes coupled to the ionization of residues buried in the hydrophobic core of a protein. The study used 25 variants of staphylococcal nuclease with buried Lys residues. The pKa values of 19 of 25 of these internal Lys residues are depressed relative to the normal pKa of Lys in water, some by as much as 5 pH units. This shift in pKa ensures that the ionizable moiety stays neutral at pH values below 10 when it is buried, consistent with what is expected from the unfavorable transfer of a charge from water to a hydrophobic environment. In most cases, ionization of the buried Lys shifts the conformation of the protein into a state where the charged Lys can contact water. This study contributes a detailed description of the location, amplitude and time scale of reorganization coupled to the ionization of buried Lys residues. The structural details of the conformational response were characterized with backbone chemical shift perturbation analysis, titrations of the buried Nζ atom, and in some cases even X-ray crystallography. At least in this protein, the anomalous pKa values of buried groups are governed by the propensity of the protein to reorganize. These results demonstrate that computational modeling of pH dependent processes in proteins will require prediction of alternative conformational states and accurate calculation of free energies; this remains a significant challenge even under the most favorable circumstances.