EXPERIMENTAL-MEASUREMENT OF THE EFFECTIVE DIELECTRIC IN THE HYDROPHOBIC CORE OF A PROTEIN

The dielectric inside a protein is a key physical determinant of the m agnitude of electrostatic interactions in proteins. We have measured t his dielectric phenomenologically, in terms of the dielectric that nee ds to be used with the Born equation in order to reproduce the observe d pK(a) shifts induced by burial of an ionizable group in the hydropho bic core of a protein. Mutants of staphylococcal nuclease with a burie d lysine residue at position 66 were engineered for this purpose. The pK(a) values of buried lysines were measured by difference potentiomet ry. The extent of coupling between the pK(a) and the global stability of the protein was evaluated by measuring pK(a) values in hyperstable forms of nuclease engineered to be 3.3 or 6.5 kcal mol(-1) more stable than the wild type. The crystallographic structure of one mutant was determined to describe the environment of the buried lysine. The diele ctrics that were measured range from 10 to 12. Published pK(a) values of buried ionizable residues in other proteins were analyzed in a simi lar fashion and the dielectrics obtained from these values are consist ent with the ones measured in nuclease. These results argue strongly a gainst the prevalent use of dielectrics of 4 or lower to describe the dielectric effect inside a protein in structure-based calculations of electrostatic energies with continuum dielectric models.