The dielectric properties of proteins are poorly understood and difficult to describe quantitatively. This limits the accuracy of methods for structure-based calculation of electrostatic energies and pKa values. The pKa values of many internal groups report apparent protein dielectric constants of 10 or higher. These values are substantially higher than the dielectric constants of 2–4 measured experimentally with dry proteins. The structural origins of these high apparent dielectric constants are not well understood. Here we report on structural and equilibrium thermodynamic studies of the effects of pH on the V66D variant of staphylococcal nuclease. In a crystal structure of this protein the neutral side chain of Asp-66 is buried in the hydrophobic core of the protein and hydrated by internal water molecules. Asp-66 titrates with a pKa value near 9. A decrease in the far UV-CD signal was observed, concomitant with ionization of this aspartic acid, and consistent with the loss of 1.5 turns of α-helix. These data suggest that the protein dielectric constant needed to reproduce the pKa value of Asp-66 with continuum electrostatics calculations is high because the dielectric constant has to capture, implicitly, the energetic consequences of the structural reorganization that are not treated explicitly in continuum calculations with static structures.
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