Critical Role of a Glycine Residue in an Allosteric Switch

Abstract

apparent that conformational reorganization coupled to the ionization of the buried group is a major determinant of these pK a values. Specifically, the creation of charge in hydrophobic environments can trigger a shift from the fully folded state to local or partially unfolded states in which the charge can gain access to water or to an environment where the charge can be solvated. These alternative conformational states are not normally populated owing to the large free energy difference between the alternative and fully-folded native states; however, the partially unfolded states can become the new ground state under pH conditions where the internal group is charged. If the ionization of an internal group promotes the transition to a new conformational state then its pK a should be sensitive to the global thermodynamic stability (DG ) of the protein because this determines the energy gap between the ground and the alternative states. This was tested by measuring the pK a of two internal Lys residues in variants of staphylococcal nuclease with thermodynamic stabilities ranging from 8.4 to 13.8 kcal/mol. The magnitude of the shift in the pK a of the internal Lys residues was found to be sensitive to the DG of the protein confirming that the pK a values of these Lys residues are determined by the probability of structural reorganization more than by local dielectric properties of their microenvironments. These observations imply that structure-based pK a calculations for buried groups and other electrostatic processes in hydrophobic environments require accurate treatment of conformational reorganization, which remains an extremely challenging proposition.