pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme

The energetics of a salt bridge formed between the side chains of aspartic acid 70 (Asp70) and histidine 31 (His31) of T4 lysozyme have been examined by nuclear magnetic resonance techniques. The pKa values of the residues in the native state are perturbed from their values in the unfolded protein such that His31 has a pKa value of 9.1 in the native state and 6.8 in the unfolded state at 10 degrees C in moderate salt. Similarly, the aspartate pKa is shifted to a value of about 0.5 in the native state from its value of 3.5-4.0 in the unfolded state. These shifts in pKa show that the salt bridge is stabilized 3-5 kcal/mol. This implies that the salt bridge stabilizes the native state by 3-5 kcal/mol as compared to the unfolded state. This is reflected in the thermodynamic stability of mutants of the protein in which Asp70, His31, or both are replaced by asparagine. These observations and consideration of the thermodynamic coupling of protonation state to folding of proteins suggest a mechanism of acid denaturation in which the unfolded state is progressively stabilized by protonation of its acid residues as pH is lowered below pH 4. The unfolded state is stabilized only if acidic groups in the folded state have lower pKa values than in the unfolded state. When the pH is sufficiently low, the acid groups of both the native and unfolded states are fully protonated, and the apparent unfolding equilibrium constant becomes pH independent. Similar arguments apply to base-induced unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic contribution of surface charge residues to the stability of a thermophilic protein: benchmarking experimental and predicted pKa values

Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01). The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81) with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.     

Electrostatic effects in unfolded staphylococcal nuclease

Structure-based calculations of pKa values and electrostatic free energies of proteins assume that electrostatic effects in the unfolded state are negligible. In light of experimental evidence showing that this assumption is invalid for many proteins, and with increasing awareness that the unfolded state is more structured and compact than previously thought, a detailed examination of electrostatic effects in unfolded proteins is warranted. Here we address this issue with structure-based calculations of electrostatic interactions in unfolded staphylococcal nuclease. The approach involves the generation of ensembles of structures representing the unfolded state, and calculation of Coulomb energies to Boltzmann weight the unfolded state ensembles. Four different structural models of the unfolded state were tested. Experimental proton binding data measured with a variant of nuclease that is unfolded under native conditions were used to establish the validity of the calculations. These calculations suggest that weak Coulomb interactions are an unavoidable property of unfolded proteins. At neutral pH, the interactions are too weak to organize the unfolded state; however, at extreme pH values, where the protein has a significant net charge, the combined action of a large number of weak repulsive interactions can lead to the expansion of the unfolded state. The calculated pKa values of ionizable groups in the unfolded state are similar but not identical to the values in small peptides in water. These studies suggest that the accuracy of structure-based calculations of electrostatic contributions to stability cannot be improved unless electrostatic effects in the unfolded state are calculated explicitly.     

Apparent pKa shifts of titratable residues at high denaturant concentration and the impact on protein stability

Urea and guanidine-hydrochloride (GdnHCl) are frequently used for protein denaturation in order to determine the Gibbs free energy of folding and kinetic folding/unfolding parameters. Constant pH value is applied in the folding/unfolding experiments at different denaturant concentrations and steady protonation state of titratable groups is assumed in the folded and unfolded protein, respectively. The apparent side-chain pKa values of Asp, Glu, His and Lys in the absence and presence of 6 M urea and GdnHCl, respectively, have been determined by 1H-NMR. pKa values of all four residues are up-shifted by 0.3-0.5 pH units in presence of 6 M urea by comparison with pKa values of the residues dissolved in water. In the presence of 6 M GdnHCl, pKa values are down-shifted by 0.2-0.3 pH units in the case of acidic and up-shifted by 0.3-0.5 pH units in the case of basic residues. Shifted pKa values in the presence of denaturant may have a pronounced effect on the outcome of the protein stability obtained from denaturant unfolding experiments.     

On the NMR analysis of pKa values in the unfolded state of proteins by extrapolation to zero denaturant

Detailed knowledge of the pH-dependence in both folded and unfolded states of proteins is essential to understand the role of electrostatics in protein stability. The increasing number of natively disordered proteins constitutes an excellent source for the NMR analysis of pKa values in the unfolded state of proteins. However, the tendency of many natively disordered proteins to aggregate via intermolecular hydrophobic clusters limits their NMR analysis over a wide pH range. To assess whether the pKa values in natively disordered polypeptides can be extrapolated from NMR measurements in the presence of denaturants, the natively disordered backbone of the C-terminal fragment 75 to 105 of Human Thioredoxin was studied. First, assignments using triple resonance experiments were performed to confirm lack of secondary structure. Then the pH-dependence of the amides and carboxylate side chains of Glu residues (Glu88, Glu95, Glu98, and Glu103) in the pH range from 2.0 to 7.0 was monitored using 2D 1H15N HSQC and 3D C(CO)NH experiments, and the behavior of their amides and corresponding carboxyl groups was compared to confirm the absence of nonlocal interactions. Lastly, the effect of increasing dimethyl urea concentration on the pKa values of these Glu residues was monitored. The results indicate that: (i) the dispersion in the pKa of carboxyl groups and the pH midpoints of amides in Glu residues is about 0.5 pH units and 0.6 pH units, respectively; (ii) the backbone amides of the Glu residues exhibit pH midpoints which are within 0.2 pH units from those of their carboxylates; (iii) the addition of denaturant produces upshifts in the pKa values of Glu residues that are nearly independent of their position in the sequence; and (iv) these upshifts show a nonlinear behavior in denaturant concentration, complicating the extrapolation to zero denaturant. Nevertheless, the relative ordering of the pKa values of Glu residues is preserved over the whole range of denaturant concentrations indicating that measurements at high denaturant concentration (e.g. 4 M dimethyl urea) can yield a qualitatively correct ranking of the pKa of these residues in natively disordered proteins whose pH-dependence cannot be monitored directly by NMR.     

Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain

The contribution of interactions involving the imidazole ring of His41 to the pH-dependent stability of the villin headpiece (HP67) N-terminal subdomain has been investigated by nuclear magnetic resonance (NMR) spin relaxation. NMR-derived backbone N-H order parameters (S2) for wild-type (WT) HP67 and H41Y HP67 indicate that reduced conformational flexibility of the N-terminal subdomain in WT HP67 is due to intramolecular interactions with the His41 imidazole ring. These interactions, together with desolvation effects, contribute to significantly depress the pKa of the buried imidazole ring in the native state. 15N R1rho relaxation dispersion data indicate that WT HP67 populates a partially folded intermediate state that is 10.9 kJ mol(-1) higher in free energy than the native state under non-denaturing conditions at neutral pH. The partially folded intermediate is characterized as having an unfolded N-terminal subdomain while the C-terminal subdomain retains a native-like fold. Although the majority of the residues in the N-terminal subdomain sample a random-coil distribution of conformations, deviations of backbone amide 1H and 15N chemical shifts from canonical random-coil values for residues within 5A of the His41 imidazole ring indicate that a significant degree of residual structure is maintained in the partially folded ensemble. The pH-dependence of exchange broadening is consistent with a linear three-state exchange model whereby unfolding of the N-terminal subdomain is coupled to titration of His41 in the partially folded intermediate with a pKa,I=5.69+/-0.07. Although maintenance of residual interactions with the imidazole ring in the unfolded N-terminal subdomain appears to reduce pKa,I compared to model histidine compounds, protonation of His41 disrupts these interactions and reduces the difference in free energy between the native state and partially folded intermediate under acidic conditions. In addition, chemical shift changes for residues Lys70-Phe76 in the C-terminal subdomain suggest that the HP67 actin binding site is disrupted upon unfolding of the N-terminal subdomain, providing a potential mechanism for regulating the villin-dependent bundling of actin filaments.     

Conformation and hydrogen ion titration of proteins: a continuum electrostatic model with conformational flexibility.

A new method for including local conformational flexibility in calculations of the hydrogen ion titration of proteins using macroscopic electrostatic models is presented. Intrinsic pKa values and electrostatic interactions between titrating sites are calculated from an ensemble of conformers in which the positions of titrating side chains are systematically varied. The method is applied to the Asp, Glu, and Tyr residues of hen lysozyme. The effects of different minimization and/or sampling protocols for both single-conformer and multi-conformer calculations are studied. For single-conformer calculations it is found that the results are sensitive to the choice of all-hydrogen versus polar-hydrogen-only atomic models and to the minimization protocol chosen. The best overall agreement of single-conformer calculations with experiment is obtained with an all-hydrogen model and either a two-step minimization process or minimization using a high dielectric constant. Multi-conformational calculations give significantly improved agreement with experiment, slightly smaller shifts between model compound pKa values and calculated intrinsic pKa values, and reduced sensitivity of the intrinsic pKa calculations to the initial details of the structure compared to single-conformer calculations. The extent of these improvements depends on the type of minimization used during the generation of conformers, with more extensive minimization giving greater improvements. The ordering of the titrations of the active-site residues, Glu-35 and Asp-52, is particularly sensitive to the minimization and sampling protocols used. The balance of strong site-site interactions in the active site suggests a need for including site-site conformational correlations.     

Effects of pH, salt, and macromolecular crowding on the stability of FK506-binding protein: an integrated experimental and theoretical study

Environmental variables can exert significant influences on the folding stability of a protein, and elucidating these influences provides insight on the determinants of protein stability. Here, experimental data on the stability of FKBP12 are reported for the effects of three environmental variables: pH, salt, and macromolecular crowding. In the pH range of 5-9, contribution to the pH dependence of the unfolding free energy from residual charge-charge interactions in the unfolded state was found to be negligible. The negligible contribution was attributed to the lack of sequentially nearest neighboring charged residues around groups that titrate in the pH range. KCl lowered the stability of FKBP12 and the E31Q/D32N double mutant at small salt concentrations but raised stability after approximately 0.5 M salt. Such a turnover behavior was accounted for by the balance of two opposing types of protein-salt interactions: the Debye-Hückel type, modeling the response of the ions to protein charges, favors the unfolded state while the Kirkwood type, accounting for the disadvantage of the ions moving toward the low-dielectric protein cavity from the bulk solvent, disfavors the unfolded state. Ficoll 70 as a crowding agent was found to have a modest effect on protein stability, in qualitative agreement with a simple model suggesting that the folded and unfolded states are nearly equally adversely affected by macromolecular crowding. For any environmental variable, it is the balance of its effects on the folded and unfolded states that determines the outcome on the folding stability.     

pK(a) values for the unfolded state under native conditions explain the pH-dependent stability of PGB1

Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK_a values. However, pK_a values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pK_a values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK_a values were determined for carboxyl groups by monitoring their pH-dependent ¹³C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pK_a values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pK_a values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK_a values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.     

pH dependent thermodynamic and amide exchange studies of the C-terminal domain of the ribosomal protein L9: implications for unfolded state structure

It is now recognized that unfolded states of globular proteins are not random coils but instead can contain significant amounts of residual structure. Here, we combine amide H/D exchange studies and thermodynamic measurements to probe pH dependent structure in the unfolded state of the small, mixed alpha-beta protein CTL9. The m value measured by urea denaturation is strongly dependent upon pD, increasing by 40% from pD 7.5 to 4.85. Likewise, the change in heat capacity upon unfolding, deltaCp(o), increases significantly from pD 7.5 to 5.5. These studies argue that the unfolded state contains interactions, presumably hydrophobic in nature, that lead to a more compact state at high pH. The expansion at lower pH correlates with the estimated unfolded state pKa values of the three histidines in CTL9 with additional contributions from acid side chains at the lower pH. Amide H/D exchange studies were conducted at pD 5.0, 6.0, and 7.0. At pD 5.0, the exchange rates could be measured for 44 residues, 29 of which exchanged by global unfolding. No evidence was found for any super protected sites, that is, sites that exchange at rates slower than those expected for global exchange. The estimated precision for the experiments limits detection to residues that are protected 2.3-fold above the intrinsic exchange rate. Thirty-seven residues could be followed at pD 6 and 27 residues at pD 7. Again no evidence for a significant super protected structure was observed. The properties of CTL9(11) are compared to other structured denatured states.     

pKa values and the pH dependent stability of the N-terminal domain of L9 as probes of electrostatic interactions in the denatured state. Differentiation between local and nonlocal interactions

pKa values were measured for the 6 carboxylates in the N-terminal domain of L9 (NTL9) by following NMR chemical shifts as a function of pH. The contribution of each carboxylate to the pH dependent stability of NTL9 was estimated by comparing the pKa values for the native and denatured state of the protein. A set of peptides with sequences derived from NTL9 were used to model the denatured state. In the protein fragments, the pKa values measured for the aspartates varied between 3.8 and 4.1 and the pKa values measured for the glutamates varied between 4.1 and 4.6. These results indicate that the local sequence can significantly influence pKa values in the denatured state and highlight the difficulties in using standard pKa values derived from small compounds. Calculations based on the measured pKa values suggest that the free energy of unfolding of NTL9 should decrease by 4.4 kcal mol-1 when the pH is lowered from 6 to 2. In contrast, urea and thermal denaturation experiments indicate that the stability of the protein decreases by only 2.6 kcal mol-1 when the carboxylates are protonated. This discrepancy indicates that the protein fragments are not a complete representation of the denatured state and that nonlocal sequence effects perturb the pKa's in the denatured state. Increasing the salt concentration from 100 to 750 mM NaCl removes the discrepancy between the stabilities derived from denaturation experiments and the stability changes calculated from the pKa values. At high concentrations of salt there is also less variation of the pKa values measured in the protein fragments. Our results argue that in the denatured state of NTL9 there are electrostatic interactions between groups both local and nonlocal in primary sequence.     

Highly perturbed pKa values in the unfolded state of hen egg white lysozyme

The majority of pK_a values in protein unfolded states are close to the amino acid model pK_a values, thus reflecting the weak intramolecular interactions present in the unfolded ensemble of most proteins. We have carried out thermal denaturation measurements on the WT and eight mutants of HEWL from pH 1.5 to pH 11.0 to examine the unfolded state pK_a values and the pH dependence of protein stability for this enzyme. The availability of accurate pK_a values for the folded state of HEWL and separate measurements of mutant-induced effects on the folded state pK_a values, allows us to estimate the pK_a values of seven acidic residues in the unfolded state of HEWL. Asp-48 and Asp-66 display pK_a values of 2.9 and 3.1 in our analysis, thus representing the most depressed unfolded state pK_a values observed to date. We observe a strong correlation between the folded state pK_a values and the unfolded state pK_a values of HEWL, thus suggesting that the unfolded state of HEWL possesses a large degree of native state characteristics.     

Linkage of proton binding to the thermal unfolding of Sso7d from the hyperthermophilic archaebacterium Sulfolobus solfataricus.

In this study the pH dependence of the thermal stability of Sso7d from Sulfolobus solfataricus is analyzed. This small globular protein of 63 residues shows a very marked dependence of thermal stability on pH: the denaturation temperature passes from 65.2 degrees C at pH 2.5 to 97.9 degrees C at pH 4.5. Analysis of the data points out that the binding of at least two protons is coupled to the thermal unfolding. By linking the proton binding to the conformational unfolding equilibrium, a thermodynamic model, which is able to describe the dependence upon the solution pH of both the excess heat capacity function and the denaturation Gibbs energy change for Sso7d, is developed. The decreased stability in very acid conditions is due to the binding of two protons on identical and noninteracting sites of the unfolded state. Actually, such sites are two carboxyl groups possessing very low pKa values in the native structure, probably involved in salt-bridges on the protein surface.     

Prediction and rationalization of the pH dependence of the activity and stability of family 11 xylanases

This paper presents a study of the pH dependence of the activity and stability of a set of family 11 xylanases for which X-ray structures are available, using the PROPKA approach. The xylanases are traditionally divided into basic and acidic xylanases, depending on whether the catalytic acid is hydrogen bonded to an Asn or Asp residue. Using X-ray structures, the predicted pH values of optimal activity of the basic xylanases are in the range of 5.2-6.9, which is in reasonable agreement with the available experimental values of 5-6.5. In the case of acidic xylanases, there are only four X-ray structures available, and using these structures, the predicted pHs of optimal activity are in the range of 4.2-5.0, compared to an observed range of 2-4.6. The influence of dynamical fluctuations of the protein structure is investigated for Bacillus agaradhaerens and Aspergillus kawachii xylanase using molecular dynamics (MD) simulations to provide snapshots from which average values can be computed. This decreases the respective predicted pH optima from 6.2-6.7 and 4.8 to 5.3 +/- 0.3 and 4.0 +/- 0.2, respectively, which are in better agreement with the observed values of 5.6 and 2, respectively. The change is primarily due to structural fluctuations of an Arg residue near the catalytic nucleophile, which lowers its pKa value compared to using the X-ray structure. The MD simulations and some X-ray structures indicate that this Arg residue can form a hydrogen bond to the catalytic base, and it is hypothesized that this hydrogen bond is stabilized by an additional hydrogen bond to another Glu residue present only in acidic xylanases. Formation of such a hydrogen bond is predicted to lower the pH optimum of A. kawachii xylanase to 2.9 +/- 0.3, which is in reasonable agreement with the observed value of 2. The predicted pH of optimal stability is in excellent agreement with the pH value at which the melting temperature (Tm) is greatest. Some correlation is observed between the pH-dependent free energy of unfolding and Tm, suggesting that the thermostability of the xylanases is partly due to a difference in residues with shifted pKa values. Thus, the thermostability of xylanases (and proteins in general) can perhaps be increased by mutations that introduce ionizable residues with pKa values significantly lower than standard values.     

Polymer models of protein stability, folding, and interactions

The unfolded state and flexible linkers in the folded structure play essential roles in protein stability and folding and protein-protein interactions. Intrinsic to these roles is the fact that unfolded proteins and flexible linkers sample many different conformations. Polymer models may capture this and complement experiments in elucidating the contributions of the unfolded state and flexible linkers. Here I review what can be predicted from these models and how well these predictions match experiments. For example, Gaussian chain models give quantitatively reasonable predictions of the effects of residual charge-charge interactions in the unfolded state and qualitatively reasonable results for the effects of spatial confinement and macromolecular crowding on protein stability. A wormlike chain model has met with success in quantifying the effects of flexible linkers in binding affinity enhancement and in regulatory switches. In future developments, more realistic models may emerge from molecular dynamics simulations, and these models will guide experiments to advance our understanding of the unfolded state and flexible linkers.     

Contribution of proton linkage to the thermodynamic stability of the major cold-shock protein of Escherichia coli CspA

The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all-beta protein unfolds reversible in a two-state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5-9.0 is linked to protonation of an amino group. This amino group appears to be the N-terminal alpha-amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute approximately 5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only approximately 1 kJ/mol. This discrepancy is related to a strong enthalpy-entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the alpha-amino group.     

Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pKa calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pKa values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pKa values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pKa values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.     

Simplified methods for pKa and acid pH-dependent stability estimation in proteins: removing dielectric and counterion boundaries

Much computational research aimed at understanding ionizable group interactions in proteins has focused on numerical solutions of the Poisson-Boltzmann (PB) equation, incorporating protein exclusion zones for solvent and counterions in a continuum model. Poor agreement with measured pKas and pH-dependent stabilities for a (protein, solvent) relative dielectric boundary of (4,80) has lead to the adoption of an intermediate (20,80) boundary. It is now shown that a simple Debye-Huckel (DH) calculation, removing both the low dielectric and counterion exclusion regions associated with protein, is equally effective in general pKa calculations. However, a broad-based discrepancy to measured pH-dependent stabilities is maintained in the absence of ionizable group interactions in the unfolded state. A simple model is introduced for these interactions, with a significantly improved match to experiment that suggests a potential utility in predicting and analyzing the acid pH-dependence of protein stability. The methods are applied to the relative pH-dependent stabilities of the pore-forming domains of colicins A and N. The results relate generally to the well-known preponderance of surface ionizable groups with solvent-mediated interactions. Although numerical PB solutions do not currently have a significant advantage for overall pKa estimations, development based on consideration of microscopic solvation energetics in tandem with the continuum model could combine the large deltapKas of a subset of ionizable groups with the overall robustness of the DH model.     

Electrostatic contributions to the stability of the GCN4 leucine zipper structure

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK_a values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK_a values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK_a values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK_a differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK_a values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK_a predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.