Structural Consequences of the Ionization of Internal Lys Residues in a Protein

Abstract

Internal ionizable residues in proteins play important functional roles in a variety of biological processes. The molecular determinants of their pKa values are poorly understood. Previously we measured the pKa of Lys, Asp, Glu and Arg at 25 internal groups in staphylococcal nuclease. 98 of these 100 variants are fully folded and native-like at pH 7. The pKa values of the majority of these groups are perturbed, some by as much as 5 pKa units, all in the direction that favors the neutral state. NMR spectroscopy was used to examine the structural and dynamic consequences of ionization of the internal lysine residues. In 9 of 10 crystal structures of Lys-containing variants the Lys side chain is completely buried, some in entirely hydrophobic microenvironments. In some cases the buried amino group makes contact with polar residues. The NMR experiments showed that in two variants the ionization of an internal Lys causes global unfolding. In five variants the ionization of the internal Lys triggers local structural changes and increased dynamics. The presence of conformational exchange in response to ionization appears to be correlated with the global stability of the variant proteins. Surprisingly, in the majority of cases, the changes in structure coupled to the ionization of the internal Lys residues are modest. These data demonstrate that proteins can tolerate internal ionizable residues, even those that exhibit large shifts in pKa values, and even in their charged states. The internal charged groups somehow manage to become solvated without disrupting the overall fold of the protein.