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

In the V23E variant of staphylococcal nuclease, Glu-23 has a p<i>K</i>_a 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 p<i>K</i>_a of an internal ionizable group can reflect the average of the very different p<i>K</i>_a values (open ≈4.5, closed ≫7.5) sampled in the different conformational states. (2) The high apparent dielectric constant reported by the p<i>K</i>_a value of internal ionizable group reflects conformational reorganization. (3) The apparent p<i>K</i>_a 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 p<i>K</i>_a values of internal residues and to reproduce pH-driven conformational transitions of proteins