Charges in Hydrophobic Environments: A Strategy for Identifying Alternative States in Proteins.

In the V23E variant of staphylococcal nuclease, Glu-23 has a pK of 7.5. At low pH, Glu-23 is neutral and buried in the hydrophobic interior of the protein. Crystal structures and NMR spectroscopy experiments show that when Glu-23 becomes charged, the protein switches into an open state in which strands β1 and β2 separate from the β-barrel; the remaining structure is unaffected. In the open state the hydrophobic interior of the protein is exposed to bulk water, allowing Glu-23 to become hydrated. This illustrates several key aspects of protein electrostatics: (1) The apparent pK of an internal ionizable group can reflect the average of the very different pK values (open ≈4.5, closed ≫7.5) sampled in the different conformational states. (2) The high apparent dielectric constant reported by the pK value of internal ionizable group reflects conformational reorganization. (3) The apparent pK of internal groups can be governed by large conformational changes. (4) A single charge buried in the hydrophobic interior of a protein is sufficient to convert what might have been a transient, partially unfolded state into the dominant state in solution. This suggests a general strategy for examining inaccessible regions of the folding landscape and for engineering conformational switches driven by small changes in pH. These data also constitute a benchmark for stringent testing of the ability of computational algorithms to predict pK values of internal residues and to reproduce pH-driven conformational transitions of proteins.