Affinity and stoichiometry of calcium binding by arsenazo III

Arsenazo III forms a 1:1 complex with calcium. The affinity constant of arsenazo III for calcium (pK_Ca) has been determined by titrating purified arsenazo III with standard calcium solutions. The method of evaluation used allows one to determine correct pK_Ca values even in the presence of micromolar amounts of contaminating calcium. The pK_Ca is influenced by the following factors: (a) in the neutral pH range the apparent pK_Ca increases strongly with pH; (b) alkali ions bind weakly to arsenazo III and millimolar concentrations cause a decrease in the apparent pK_Ca; (c) the magnesium affinity of arsenazo III, although much lower than the calcium affinity, increases strongly with pH in the neutral range (at pH 7.0 the calcium affinity of arsenazo III is not appreciably altered by up to 2 mm magnesium); (d) strontium and barium form weaker complexes with arsenazo III than calcium, but much stronger complexes than magnesium; (e) the apparent pK_Ca decreases with increasing buffer concentration in the millimolar range. The pK_Ca of arsenazo III is so high that, unless the arsenazo III concentration greatly exceeds the calcium concentration, a considerable fraction of the total arsenazo III is in the calcium complexed form. Because of this, arsenazo III responds nonlinearly to all but the lowest calcium concentrations; however, quantitation of the calcium concentration can readily be done from the mass action law provided that the pK_Ca is determined under the actual experimental conditions. Arsenazo III is a reliable calcium indicator if the experimental conditions, particularly pH, are well controlled.     

Isocitrate lyase from Pseudomonas indigofera: pH dependence of catalysis and binding of substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lysase from Pseudomonas indigofera was dependent on two dissociable groups (pK_a's of 6.9 and 8.6). The pH dependence of the pK_i for succinate, a product of isocitrate cleavage, implied that a dissociable group (pK_a of 6.0) on the enzyme functions in binding succinate. The pK_i's for maleate and itaconate (succinate analogs) were similarly pH dependent. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, was pH independent. In contrast the pK_i's of the four-carbon dicarboxylic acid inhibitors, fumarate and meso-tartrate, both of which affect the glyoxylate site, were dependent on a dissociable group on the enzyme-inhibitor complex. Comparison of the pH dependence of the pK_m for isocitrate and the pK_i for succinate (and succinate analogs) indicated that the binding of isocitrate was dependent on an acidic dissociable group on the enzyme (pK_a of 5.8). The pH dependence of the pK_i for homoisocitrate was similar. In addition the K_i for succinate and K_m for isocitrate were dependent upon Mg²⁺ concentration. Inhibition by phosphoenolpyruvate, which binds to the succinate site and may regulate isocitrate lyase from P. indigofera, was twice as pH dependent as that for succinate. Two dissociable groups, one on the enzyme (pK_a of 5.8) and one on phosphoenolpyruvate (pK_a of 6.35), contributed to the pH dependence observed with phosphoenolpyruvate.     

pH dependence of conformational fluctuations of the protein backbone

Proton binding equilibria (pK_a values) of ionizable groups in proteins are exquisitely sensitive to their microenvironments. Apparent pK_a values measured for individual ionizable residues with NMR spectroscopy are actually population‐weighted averages of the pK_a in different conformational microstates. NMR spectroscopy experiments with staphylococcal nuclease were used to test the hypothesis that pK_a values of surface Glu and Asp residues are affected by pH‐sensitive fluctuations of the backbone between folded and locally unfolded conformations. ¹⁵N spin relaxation studies showed that as the pH decreases from the neutral into the acidic range the amplitudes of backbone fluctuations in the ps‐ns timescale increase near carboxylic residues. Hydrogen exchange experiments suggested that backbone conformational fluctuations promoted by decreasing pH also reflect slower local or sub‐global unfolding near carboxylic groups. This study has implications for structure‐based pK_a calculations: (1) The timescale of the backbone's response to ionization events in proteins can range from ps to ms, and even longer; (2) pH‐sensitive fluctuations of the backbone can be localized to both the segment the ionizable residue is attached to or the one that occludes the ionizable group; (3) Structural perturbations are not necessarily propagated through Coulomb interactions; instead, local fluctuations appear to be coupled through the co‐operativity inherent to elements of secondary structure and to networks of hydrogen bonds. These results are consistent with the idea that local conformational fluctuations and stabilities are important determinants of apparent pK_a values of ionizable residues in proteins. Proteins 2014; 82:3132–3143. © 2014 Wiley Periodicals, Inc.     

Heme-linked ionization and chloride binding in intestinal peroxidase and lactoperoxidase.

Hog intestinal peroxidase and bovine lactoperoxidase exhibited similar spectral shifts upon pH alteration. From spectrophotometric titrations, it was found that there are hemelinked ionizations of pK_a = 4.75 in intestinal peroxidase and pK_a = 3.5 in lactoperoxidase. The apparent pK_a (pK_a′) increased with the increase in chloride concentration. The pK_a′ vs log[Cl^?] plots showed that the chloride forms complex with the acid forms of these enzymes with a dissociation constant (pK = 2.7). Although the dissociation constant (K_d) of the peroxidase-cyanide complexes is nearly independent of pH, cyanide competed with chloride in the acidic pH region. The slopes of logK_d vs log[Cl^?] were 1.0 for intestinal peroxidase and 0.5 for lactoperoxidase. The reaction of hydrogen peroxide with these peroxidases was also affected by chloride, similarly as the reaction with cyanide was. The results were explained by assuming that protonation occurs at the distal base and destroys the hydrogen bond between the base and a water molecule at the sixth coordinate position of the heme iron.     

Effect of ionic strength on the pKa of ligands bound to DNA

The pK_a of 3,8-diamino-6-phenyl-phenanthridine (DAPP), a nonquaternary analog of ethidium bromide, has been determined spectrophotometrically as a function of sodium ion concentration both free in solution and complexed to DNA. Unwinding angle determinations with this compound were determined with Col El DNA using ethidium bromide as a standard. The unwinding angle for DAPP was 24 ± 2° relative to 26° for ethidium, and this suggests that DAPP binds in a manner quite similar to ethidium and with no significant outside bound DAPP under these experimental conditions. Isobestic behavior was obtained on spectrophotometric pH titration above pH 5 as long as the ratio of DNA-phosphate to ligand was between 100 and 300 and the DNA phosphate concentration was approximately 0.01M or greater. The loss of isosbestic behavior which occurred below pH 5 is probably due to titration of the 8 amino group of the ligand complexed to DNA. To circumvent this problem, pK_a values and the extinction coefficient of the acidic species were both determined by a computer program using experimental data obtained above pH 5. The pK_a of the free compound has only a minor dependence on ionic strength, while the pK_a of the ligand bound to DNA in an intercalated complex depends strongly on the sodium ion concentration. The pK_a of the DAPP-DNA complex is a linear function of –log[Na⁺] as predicted by the ion-condensation theory of polyelectrolytes. It was determined that DAPP is essentially completely bound to DNA under the conditions of these experiments by (1) determination of apparent pK_a values as a function of total DNA concentration, (2) calculation of binding constants for the neutral species of DAPP, and (3) spectral analysis of the protonated and neutral species of DAPP bound to DNA relative to DAPP free in solution. These results support the ion-condensation theory; provide an independent method for measuring ψ*, the average number of counterions associated per phosphate of DNA in the intercalated conformation; and illustrate that there are no specific pH effects or absolute pK_a values for ligands bound to DNA, but only ionic-strength-dependent results.     

Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations

Multi-conformation continuum electrostatics (MCCE) was used to analyze various structures of the NS3 RNA helicase from the hepatitis C virus in order to determine the ionization state of amino acid side chains and their pK_as. In MCCE analyses of HCV helicase structures that lacked ligands, several active site residues were identified to have perturbed pK_as in both the nucleic acid binding site and in the distant ATP-binding site, which regulates helicase movement. In all HCV helicase structures, Glu493 was unusually basic and His369 was abnormally acidic. Both these residues are part of the HCV helicase nucleic acid binding site, and their roles were analyzed by examining the pH profiles of site-directed mutants. Data support the accuracy of MCCE predicted pK_a values, and reveal that Glu493 is critical for low pH enzyme activation. Several key residues, which were previously shown to be involved in helicase-catalyzed ATP hydrolysis, were also identified to have perturbed pK_as including Lys210 in the Walker-A motif and the DExD/H-box motif residues Asp290 and His293. When DNA was present in the structure, the calculated pK_as shifted for both Lys210 and Asp290, demonstrating how DNA binding might lead to electrostatic changes that stimulate ATP hydrolysis.     

Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase

Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp³⁰⁹ in substrate binding and catalysis. These studies predicted an elevated pK_a for Asp³⁰⁹ and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp³⁰⁹ with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp³⁰⁹ based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pK_a of 8.2 observed for androstenedione binding to the side chain of Asp³⁰⁹. To our knowledge, this work represents the first experimental assignment of a pK_a value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7–8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures.     

Urea-induced modification of cytochrome c flexibility as probed by cyanide binding

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant k_a increased 20-fold when the concentration of urea was raised from 0 M to 6 M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the pK of acid-induced unfolding, the k_a value reached its maximum (k_a,max) in all urea concentrations. Interestingly, the k_a,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe–S(Met80) bond disruption and for ligand binding to heme iron.     

Synthesis of antibacterial pseudopeptides with less hemolytic activity from a cytotoxic peptide and their pH-dependent activity

We synthesized antibacterial pseudopeptides with less hemolytic activity by incorporation of reduced amide bond ψ[CH₂NH] into α helical antibacterial peptide with hemolytic activity. As the pK_a value of reduced amide bond is 7–8, it is protonated depending on the pH. We investigated the secondary structure, the binding affinity and the leakage activity for the vesicles, and the antibacterial activity of the peptide and its pseudopeptides at neutral and basic pH. Unlike the peptide, the pseudopeptides showed a more potent leakage activity when pH increased. The peptide exhibited a lower antibacterial activity at basic pH than at neutral pH, whereas the pseudopeptide showed the same antibacterial activity at basic and neutral pH. Overall results indicated that hydrophobicity of backbone of the pseudopeptide plays an important role in the increase of leakage activity and retention of antibacterial activity at basic pH.     

Electrostatic control of enzyme reactions: The mechanism of inhibition of glucose oxidase by putrescine

The interaction of putrescine dihydrochloride with glucose oxidase is reported. At pH 7.65 glucose oxidase is strongly anionic (Z = ?80). The pK_a of an essential acidic group on the reduced form of the enzyme is extremely sensitive to ionic strength, as predicted by simple electrostatic theory [J. G. Voet, J. Coe, J. Epstein, V. Matossian, and T. Shipley (1981), Biochemistry, 20, 7182–7185]. Putrescine dihydrochloride was found to inhibit glucose oxidase at pH 7.65 at a constant ionic strength of 0.05. The kinetics do not obey simple competitive inhibition, however. The data can best be explained by a model in which change in the electrostatic potential of the enzyme on putrescine binding changes the observed pK_a of the essential acidic group. The pH dependence of putrescine inhibition supports this interpretation. At I = 0.05, 5 mM putrescine was found to change the pK_a of the essential acidic group from 7.6 to 7.1. The shift in the pK_a as a function of putrescine concentration at pH 7.7 and I = 0.05 also supports the model presented. The K_a for putrescine to the active form of the enzyme was calculated to be 4.2 mm.     

Partial purification and characterization of cyclic AMP phosphodiesterases from Funaria hygrometrica

Natural-abundance ¹³C NMR spectroscopy at 15.04 MHz has been used to examine the effects of pH, calcium, and lanthanide ions on the polypeptide cardiac stimulant Anthopleurin-A in aqueous solution. The carboxyl resonance from the aspartic acid residue not observed in a previous study (R. S. Norton and T. R. Norton, 1979, J. Biol. Chem.254, 10220–10226) has been identified and an apparent pK_a of 3.4 obtained. More accurate estimates have been derived for the apparent pK_a values of the two histidine residues. Binding of Ca²⁺ ions has been found by equilibrium dialysis and ¹³C NMR to be weak (K_d > 0.1 M). The interaction with lanthanide ions is slightly stronger, but binding occurs at the C terminus as well as at a site involving one or both of the aspartate carboxylate groups. These results suggest that possible Anthopleurin-A-induced calcium translocation in the myocardial cell is a secondary effect. The interaction of Anthopleurin-A with lipid monolayers has also been examined. Binding occurs to neutral and zwitterionic lipids, but is stronger with negatively charged lipids, particularly cardiolipin. This interaction is also influenced by the presence of Ca²⁺ ions. The implications of these results for the mechanism of action of this polypeptide are discussed.     

The deacylation of substituted benzol-alpha-chymotrypsins.

An extensive comparison of deacylation rates of mono- and disubstituted benzoyl-α-chymotrypsins indicates that no steric effects on rate or apparent pK_a of deacylation are detectable within this series. Some anomalous effects on deacylation rate appear to be associated with fluoro- and nitro-substituents in particular positions on the ring and may be attributable to specific interactions at the enzyme active site. The extensive series of structurally similar acyl-enzymes prepared has allowed a thorough analysis of the effect of acyl group pK_a on the apparent pK_a of deacylation. The data indicates that polar effects on the apparent pK_a are probably negligible. Rho for the deacylation reaction is in good agreement with model reactions for an imidazole general base-catalyzed model reaction.     

Arginine: Its pKa value revisited

Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pK_a value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pK_a value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pK_a value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.     

Surface potential and energy-coupling in bioenergy-conserving membrane systems

In order to understand the localization of dyes and the nature of their responses in membranes and particularly in those involved in energy-conservation processes, the influence of micelles of neutral and ionic surfactants on the pK _a of solubilized fluorophoric (umbelliferone) and chromophoric (bromthymol blue and methyl red) indicator dyes is studied. It is shown that the pK _a of the indicator adsorbed onto micelles shifted towards the acid extreme with cationic micelles, to the alkaline side with anionic micelles while it was not significantly modified by the neutral ones. Maximal displacements were observed with Methyl Red where the difference in pK _a between anionic and cationic micelles was as large as 3 pH units. Phospholipid liquid crystals (Liposomes) of phosphatidylcholine with and without adsorbed long-chain ions introduced in order to confer to it a net surface charge induced displacements of the pK _a of UBF analogous to those detected in the presence of detergent micelles. It was demonstrated that UBF can monitor reversal of charge phenomena such as that obtained by the interaction of phosphatidylcholine + dicetyl phosphate liposomes (anionic colloid) with poly-L-lysine (cationic colloid). The partition of the indicator dyes between micellar and aqueous phases was determined by gel filtration revealing thequasi exclusive presence of the dyes in the micellar phase. Fluorescence polarization measurement of solubilized UBF in either ionic micelles or submitochondrial particles indicate that the dye tumbling rate is as rapid as in pure water suggesting that the dye is mobile in an interfacial environment where it can experience modifications due to changes in surface potential. The use of UBF as a probe of respiration-dependent energy-linked reactions in submitochondrial particles is presented. The available data on the use of indicator dyes in mitochondrial, chloroplast and bacterial chromatophore membranes is reevaluated, on the basis of the evidence of the extreme sensitivity of these probes to surface charge. The implications of these results and considerations are discussed in terms of the importance of the surface potential in the primary event of the energy-coupling process in oxidative and photosynthetic phosphorylation.A preliminary account of this research has been presented elsewhere (IV International Biophysics Congress of the International Union of Pure and Applied Biophysics, Moscow, August 1972).     

Isocitrate lyase from Neurospora crassa: pH dependence of catalysis and interaction with substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lyase from Neurospora crassa is dependent on two dissociable groups with pK_a values of 6.1 and 8.6. A dissociable group with a pK_a of 8.5 on the enzyme-substrate complex affects the pK_m for isocitrate. The pK_i for homoisocitrate is affected in a like manner. The pH dependence of the pK_i's for succinate, a product of isocitrate cleavage, and the succinate analog maleate is similar to the pH dependence of the pK_m of isocitrate below pH 7.3, but is markedly different above this pH. Both the K_m for isocitrate and the K_i for succinate were dependent upon Mg²⁺ concentration. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, is dependent on a group with a pK_a of 6.8 on the enzyme-inhibitor complex. The pH dependence of the pK_i for phosphoenolpyruvate, which binds to the succinate site, suggests that it is dependent on two dissociable groups, one on phosphoenolpyruvate and one, by analogy to the pK_m for isocitrate, on the enzyme-glyoxylate-inhibitor complex.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

Ionisations within a subtilisin–glyoxal inhibitor complex

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K_i=2.3±0.2 μM at pH 7.0 and 25 °C. Using Z-Ala-Pro-[2-¹³C]Phe-glyoxal we have detected a signal at 107.3 ppm by ¹³C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the ¹³C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK_a<3. This is the first observation of oxyanion formation in a reversible subtilisin–inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK_a of ～6.5 in the free enzyme and on a pK_a<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol^?1. We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.     

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei

The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pK_a of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. Wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pK_a 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pK_a, 7.17 and 9.64, in the enzyme–substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pK_a from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pK_a from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutant enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈ 6.1 kcal/mol. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240–325 cal/mol. However proton balance evidences the dissociation of other group(s) that can be collectively described by a single pK_a shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2–2.3 kcal/mol to set the enzyme into a catalytically competent form.     

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK_a, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK_a is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab₂ antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.     

Structural origins of high apparent dielectric constants experienced by ionizable groups in the hydrophobic core of a protein

The side chains of Lys66, Asp66, and Glu66 in staphylococcal nuclease are fully buried and surrounded mainly by hydrophobic matter, except for internal water molecules associated with carboxylic oxygen atoms. These ionizable side chains titrate with pK_a values of 5.7, 8.8, and 8.9, respectively. To reproduce these pK_a values with continuum electrostatics calculations, we treated the protein with high dielectric constants. We have examined the structural origins of these high apparent dielectric constants by using NMR spectroscopy to characterize the structural response to the ionization of these internal side chains. Substitution of Val66 with Lys66 and Asp66 led to increased conformational fluctuations of the microenvironments surrounding these groups, even under pH conditions where Lys66 and Asp66 are neutral. When Lys66, Asp66, and Glu66 are charged, the proteins remain almost fully folded, but resonances for a few backbone amides adjacent to the internal ionizable residues are broadened. This suggests that the ionization of the internal groups promotes a local increase in dynamics on the intermediate timescale, consistent with either partial unfolding or increased backbone fluctuations of helix 1 near residue 66, or, less likely, with increased fluctuations of the charged side chains at position 66. These experiments confirm that the high apparent dielectric constants reported by internal Lys66, Asp66, and Glu66 reflect localized changes in conformational fluctuations without incurring detectable global structural reorganization. To improve structure-based pK_a calculations in proteins, we will need to learn how to treat this coupling between ionization of internal groups and local changes in conformational fluctuations explicitly.