Prior computational studies of the acid-unfolding behavior of staphylococcal nuclease (SNase) suggest that the pKa values of its carboxylic groups are difficult to reproduce with electrostatics calculations with continuum methods. To examine the molecular determinants of the pKa values of carboxylic groups in SNase, the pKa values of all 20 Asp and Glu residues were measured with multidimensional and multinuclear NMR spectroscopy in an acid insensitive variant of SNase. The crystal structure of the protein was obtained to describe the microenvironments of the carboxylic groups. Fourteen Asp and Glu residues titrate with relatively normal pKa values that are depressed by less than 1.1 units relative to the normal pKa of Asp and Glu in water. Only six residues have pKa values shifted by more than 1.5 units. Asp-21 has an unusually high pKa of 6.5, which is probably the result of interactions with other carboxylic groups at the active site. The most perturbed pKa values appear to be governed by hydrogen bonding and not by Coulomb interactions. The pKa values calculated with standard continuum electrostatics methods applied to static structures are more depressed than the measured values because Coulomb effects are exaggerated in the calculations. The problems persist even when the protein is treated with the dielectric constant of water. This can be interpreted to imply that structural relaxation is an important determinant of the pKa values; however, no major pH-sensitive conformational reorganization of the backbone was detected using NMR spectroscopy. Proteins 2009. © 2009 Wiley-Liss, Inc.
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