Experimental pKa 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., Biophys. J. 79:1610-1620; García-Moreno E. et al., 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 (epsilon(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 epsilon(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, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.

High tolerance for ionizable residues in the hydrophobic interior of proteins

“…1B) or, second, by direct measurement of the H ϩ titration curves of the internal Lys residue (Fig. 1C) (11)(12)(13)22). These 2 methods are equivalent, as shown by the simulations in Fig.…”

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

“…For clarity, the transferred proton is shown in green and the D103-phenol hydrogen bond has been omitted. lations (3)(4)(5). Our study provides an example in which a more controlled system with minimal structural rearrangements and incisive knowledge of ionization states may more cleanly isolate electrostatic effects and substantially improve computational accuracy.…”

“…Furthermore, it remains extremely challenging to study the electrostatic consequences of charge rearrangements that accompany hydrogen bond-mediated proton transfers. Few experimental methods exist to vary the ionization properties of discrete protein groups incrementally, and structural rearrangements within the protein matrix that typically accompany charge rearrangements complicate computational modeling and the straightforward interpretation of the electrostatic properties of protein active sites and interiors (3)(4)(5).…”

Computational Methods for Biomolecular Electrostatics

“…The correct prediction of protein titration states is important for the analysis of enzyme mechanisms, protein stability, and molecular recognition. As mentioned earlier, efforts have been underway for more than 80 years (Linderström-Lang, 1924;Antosiewicz et al, 1996b;Bastyns et al, 1996;Luo et al, 1998;Nielsen and Vriend, 2001;Fitch et al, 2002;Georgescu et al, 2002;Li et al, 2002;Alexov, 2003;Nielsen and McCammon, 2003;Li et al, 2004;Jensen et al, 2005;Krieger et al, in press) to correctly predict protein titration states and understand the determinants of pK a s for amino acids in protein environments (see chapter by Whitten, et al in this volume).…”