Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

Autor: C. Nick Pace, D Schell, James M. Briggs, Stephanie Newsom, Richard L. Thurlkill, Saul R. Trevino, Marta Bruix, Gerald R. Grimsley, Jan M. Antosiewicz, Kevin L. Shaw, Beatrice M. P. Huyghues-Despointes, Douglas V. Laurents, J. Martin Scholtz, Manuel Rico
Přispěvatelé: National Institutes of Health (US), Welch Foundation, Dirección General de Investigación Científica y Técnica, DGICT (España)
Rok vydání: 2003
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Zdroj: Digital.CSIC. Repositorio Institucional del CSIC
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Popis: The pK 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 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge–dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds. A primary objective in this study was to investigate the influence of charge–charge interactions on the pK 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 pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge–charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pKSa−pK5K) 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 pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK 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 pK 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 pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.
This work was supported by grants GM-37039 and GM-52483 from the National Institutes of Health (USA), grants BE-1060 and BE-1281 from the Robert A. Welch Foundation, and a grant PB-93-06777 to M.R. from the Dirección General de Investigación Cientı́fica y Técnica (Spain)
Databáze: OpenAIRE
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