Structural and energetic studies of the effects of temperature and dehydration on proton binding equilibria in proteins

Nearly all pH- and salt-dependent phenomena in biological macromolecules can be attributed to electrostatic interactions. The charged state of an ionizable group can be affected by charge-charge interactions with other ionizable groups, charge-dipole interactions with the solvent or dipoles within the protein, or other external variables such as temperature. Although charge-charge interactions can be accurately described, the effects of dehydration and the dielectric response of the protein on ionization behavior are not understood completely. This thesis explores the effect of temperature and the internal dielectric response of the protein on the ionization behavior of titratable groups in proteins.; The ability to make structure-based predictions about the electrostatic contributions to proton binding, heat capacity, and thermostability would allow these effects to be explored at the microscopic level. The solvent accessibility modified Tanford-Kirkwood algorithm was modified to include the effects of temperature on the ionization of titratable groups. The hydrogen ion titration curves of sperm whale myoglobin and horse cytochrome c were measured potentiometrically over a broad pH and temperature range and these results were compared to titration curves predicted by the model. The enthalpy of ionization of titratable groups and the temperature dependence of the external dielectric constant were shown to accurately describe the observed temperature dependence of the titration curves.; The pKa values of different ionizable groups buried within the hydrophobic core of staphylococcal nuclease were determined by difference potentiometry. From the energetics of these groups, the effective dielectric constant of the protein interior was determined to be 10-15. The high resolution x-ray crystallographic structure of a mutant of staphylococcal nuclease where valine at position 66 has been replaced by glutamic acid contains a linear arrangement of four highly ordered solvent molecules that lead from the carboxyl oxygens of E66 to the bulk solvent. The observation of solvent penetration for this group provides a physical basis for the use of a higher dielectric constant in theoretical models that predict electrostatic phenomena.