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 p K 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 T 1 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. Proteins 2002;46:85–96. © 2001 Wiley‐Liss, Inc.