Site-specific contributions to the pH dependence of protein stability

Abstract

Understanding protein stability is a significant challenge requiring characterization of interactions within both folded and unfolded states. Of these, electrostatic interactions influence ionization equilibria of acidic and basic groups and diversify their pK a values. The pH dependence of the thermodynamic stability (⌬G FU) of a protein arises as a consequence of differential pK a values between folded and unfolded states. Previous attempts to calculate pHdependent contributions to stability have been limited by the lack of experimental unfolded state pK a values. Using recently developed NMR spectroscopic methods, we have determined residuespecific pK a values for a thermodynamically unstable Src homology 3 domain in both states, enabling the calculation of the pH dependence of stability based on simple analytical expressions. The calculated pH stability profile obtained agrees very well with experiment, unlike profiles derived from two current models of electrostatic interactions within unfolded states. Most importantly, per-residue contributions to the pH dependence of ⌬G FU derived from the data provide insights into specific electrostatic interactions in both the folded and unfolded states and their roles in protein stability. These interactions include a hydrogen bond between the Asp-8 side-chain and the Lys-21 backbone amide group in the folded state, which represents a highly conserved interaction in Src homology 3 domains.

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

“…Furthermore they demonstrated that the non-native interactions persisted in the transition state for folding [18,31]. DSE effects involving charged residues have also been detected in the drkN SH3 domain [16], although they are believed to be due interactions involving adjacent residues. Work on T4 lysozyme, RNase A and RNase T1 have shown that mutations that are predicted to increase protein stability by altering native state electrostatic interactions often have much smaller effects than expected.…”

“…However, indirect methods normally need to be used to study the DSE under native conditions [13]. A wide range of approaches have been used including, but not limited to, the study of peptide fragments, the analysis of destabilizing point mutants or truncation mutants, amide 1 H/ 2 H exchange experiments of the native state, changes in m-values upon mutation and the analysis of the pH dependence of protein stability [14][15][16][17]. Numerous investigations have provided compelling evidence for compact DSEs with significant amounts of residual structure under native conditions.…”

Electrostatic Contributions to the Stability of the GCN4 Leucine Zipper Structure

“…For these reasons, pK a values obtained directly from the D state are more accurate than those inferred from random coil model compounds 12,28,29,35 . To model the D state of GCN4p we studied the protein in 6 M urea, since this is often the endpoint of protein denaturation studies 13 .…”

A summary of the measured pK values of the ionizable groups in folded proteins

“…Most of these pKs do not differ significantly from the intrinsic pK values estimated earlier by Nozaki and Tanford. 2 It will be interesting to compare these values to the pKs determined in unfolded proteins. [3][4][5][6][7][8][9][10] Most of the pK values for folded proteins have been measured directly using techniques based on nuclear magnetic resonance (NMR). [11][12][13] A smaller number have been measured using indirect techniques.…”