Continuum electrostatic analysis of irregular ionization and proton allocation in proteins.

Irregular (nonsigmoidal) ionization behavior of titratable groups in proteins is analyzed theoretically, using a computational algorithm designed to count explicitly for tautomers of titratable groups and different locations of polar hydrogens. On the basis of calculations for different model systems (acid-acid, base-base, acid-base pairs, and cluster of three strongly interacting groups), it is demonstrated that the pK values, extracted from nonsigmoidal titration curves by fitting to a sum of Henderson-Hasselbalch equations, do not describe the ionization equilibrium correctly. The conditions for observation of irregular titration curves are derived analytically for the case of arbitrary couple of interacting ionizable groups. A possible relation between irregularly shaped titration curves and tautomerization is also illustrated. The protonation-deprotonation equilibrium of Asp76 in ribonuclease T1 is shown to be coupled to dipole reorientation of a water molecule bound at the protein-solvent interface. This finding provides a new interpretation of the experimentally observed chemical shift of this residue.