Charge–Charge Interactions are Key Determinants of the p K Values of Ionizable Groups in Ribonuclease Sa (pI=3.5) and a Basic Variant (pI=10.2)

Abstract

The p K values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 °C in 0.1 M NaCl. In RNase Sa, 18 p K values and in 5K, 11 p K values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest p K (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated p K (7.4). These results highlight the importance of desolvation and charge–dipole interactions in perturbing p K values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased p K values relative to model compounds. A primary objective in this study was to investigate the influence of charge–charge interactions on the p K values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the α-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge–dipole interactions will have comparable effects on the p K values of both. Their p K differences, therefore, are expected to be chiefly due to the different charge–charge interactions. As anticipated from its higher net charge, all measured p K values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The p K differences (p K Sa−p K 5K) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the p K differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. p K values calculated using Coulomb's Law also showed a good correlation ( R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson–Boltzmann equation, which considers desolvation and charge–dipole interactions in addition to charge–charge interactions, was also used to calculate p K values. Surprisingly, these values are more poorly correlated ( R=0.65) with the values from experiment. Taken together, the results are evidence that charge–charge interactions are the chief perturbant of the p K values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.