Engineering of Artificial pH Switch Proteins using Internal Ionizable Residues with Anomalous PKA Values

Abstract

Many proteins act as pH sensors. They can respond to small changes in pH with a large conformational transition of functional significance. The thermodynamic basis of pH-driven conformational changes in proteins is well understood: a change in pH can drive a conformational transition when final and initial states have different affinity for H+. This requires that some ionizable groups titrate with different pKa values in the two states. Here we demonstrate how buried Lys or Glu residues with highly anomalous pKa values can be used for rational engineering of artificial pH switch protein that undergo global, cooperative unfolding in response to small changes in pH in the physiological range. This study was enabled by our previous demonstration that most Lys and Glu residues buried in the interior of staphylococcal nuclease titrate with highly anomalous pKa values as a consequence of being buried in the hydrophobic interior of the protein. The ionization of a single buried ionizable group is not sufficient to drive the unfolding of the protein, thus we engineered variants with two buried ionizable groups. The V66E/A109E and T62K/L125K variants of SNase were selected because, based on the pKa values measured for these buried residues, the protein was expected to act as a switch near pH 7. This was confirmed with equilibrium thermodynamic experiments. The double Glu variant was stable at low pH and unfolded at high pH and the double Lys variant was stable at high pH and unfolded at low pH, as expected from the nature of the shifts in pKa values. Crystallography and NMR spectroscopy were used to characterize the folded and unfolded states. This strategy for the design of artificial pH sensing proteins is completely general and transferable to other proteins.