Experimental pK a Values of Buried Residues: Analysis with Continuum Methods and Role of Water Penetration

Abstract

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK a values of 5.7 and 8.8, respectively (Dwyer et al., 2000, Biophys. J. 79:1610–1620; García-Moreno E. et al., 1997, Biophys. Chem. 64:211–224). Continuum calculations with static structures reproduced the pK a values when the protein interior was treated with a dielectric constant ( ϵ in) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK a calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK a values of buried residues were reproduced with ϵ in in the range 4–5. The calculations show that internal waters can modulate pK a values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, ϵ in of 5–7 reproduced the pK a values. These values of ϵ in > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.