The conserved, buried aspartic acid in oxidized Escherichia coli thioredoxin has a pKa of 7.5. Its titration produces a related shift in global stability.

Aspartic acid 26 in Escherichia coli thioredoxin is located at the bottom of a hydrophobic cavity, near the redox-active disulfide of the active site. Asp 26 is embedded in the protein except for part of the surface of one carboxyl oxygen. The high degree of evolutionary conversion of Asp 26 suggests that it plays a critical role in thioredoxin function. We have determined the pKa of Asp 26 by a novel electrophoretic method based on the relative electrophoretic mobilities of wild-type thioredoxin and of D26A thioredoxin (with Asp 26 replaced by alanine). The pKa of Asp 26 determined by this technique is 7.5, more than 3 units above the pKa of a solvated carboxyl side chain. The titration of Asp 26 is thermodynamically linked to the stability of thioredoxin. As expected if thioredoxin stability depends on the ionization state of Asp 26, delta Go WT, the free energy of the cooperative denaturation reaction of wild-type thioredoxin by guanidine hydrochloride, varies with pH in a sigmoidal fashion in the vicinity of pH 7.5. Over the same pH range, the free energy for D26A folding, delta Go D26A, is pH independent and D26A is highly stabilized compared to wild type. From the thermodynamic cycle describing the linkage of Asp 26 titration to thioredoxin stability, the difference in free energy between wild-type thioredoxin with protonated Asp 26 and wild-type thioredoxin with deprotonated Asp 26, delta delta Go (COOH----COO-), is calculated to be 4.9 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.     

Proton-transfer effects in the active-site region of Escherichia coli thioredoxin using two-dimensional 1H NMR.

A series of two-dimensional (2D) correlated 1H NMR spectra of reduced and oxidized Escherichia coli thioredoxin have been used to probe the effects of pH in the vicinity of the active site, -Cys32-Gly-Pro-Cys35-, using the complete proton resonance assignments available for thioredoxin. In either oxidation state, the majority of residues of the thioredoxin molecule remain unchanged between pH 5.7 and pH 10, as indicated by the identical chemical shifts of the C alpha H, C beta H, and other protons. In reduced thioredoxin, a fairly widespread region around the active-site dithiol is affected by the titration of a group or groups with pKa approximately 7.1-7.4 in 2H2O. Another titration, with pKa approximately 8.4, affects a smaller region of the protein. Oxidized thioredoxin contains a disulfide and no free thiol groups; nevertheless, the proton resonances of many groups in the active-site region were observed to titrate with a pKa of 7.5, probably as a result of an abnormally high pKa value for the carboxyl group of the buried Asp-26 residue. For reduced thioredoxin, the results indicate that Asp-26 is titrating in this pH range, as well as both thiol groups. The new results are strongly suggestive that the mechanism of thioredoxin-catalyzed protein disulfide reduction may be critically dependent on proton transfer as well as electron transfer within the active site.     

Escherichia coli thioredoxin inhibition by cadmium: two mutually exclusive binding sites involving Cys32 and Asp26.

Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd(2+) led us to investigate the thioredoxin-cadmium interaction properties. We used calorimetric and spectroscopic methods at different pH values to explore the relative contribution of putative binding residues (Cys32, Cys35, Trp28, Trp31 and Asp26) within or near the active site. At pH 8 or 7.5 two binding sites were identified by isothermal titration calorimetry with affinity constants of 10 x 10(6) m(-1) and 1 x 10(6) m(-1). For both sites, a proton was released upon Cd(2+) binding. One mole of Cd(2+) per mole of reduced thioredoxin was measured by mass spectrometry at these pH values, demonstrating that the two binding sites were partially occupied and mutually exclusive. Cd(2+) binding at either site totally inhibited the thiol-disulfide transferase activity of Trx. The absence of Cd(2+) interaction detected for oxidized or alkylated Trx and the inhibition of the enzymatic activity of thioredoxin by Cd(2+) supported the role of Cys32 at the first site. The fluorescence profile of Cd(2+)-bound thioredoxin differed, however, from that of oxidized thioredoxin, indicating that Cd(2+) was not coordinated with Cys32 and Cys35. From FTIR spectroscopy, we inferred that the second site might involve Asp26, a buried residue that deprotonates at a rather high and unusual pK(a) for a carboxylate (7.5/9.2). The pK(a) of the two residues Cys32 and Asp26 have been shown to be interdependent [Chivers, T. P. (1997) Biochemistry36, 14985-14991]. A mechanism is proposed in which Cd(2+) binding at the solvent-accessible thiolate group of Cys32 induces a decrease of the pK(a) of Asp26 and its deprotonation. Conversely, interaction between the carboxylate group of Asp26 and Cd(2+) at a second binding site induces Cys32 deprotonation and thioredoxin inhibition, so that Cd(2+) inhibits thioredoxin activity not only by binding at the Cys32 but also by interacting with Asp26.     

pH dependence of stability of the 10th human fibronectin type III domain: a computational study

We present detailed computational studies based on electrostatic calculations to evaluate the origins of pKa values and the pH dependence of stability for the 10th type III domain of human fibronectin (FNfn10). One of our goals is to validate the calculation protocols by comparison to experimental data (Koide, A.; Jordan, M. R.; Horner, S.; Batori, V.; Koide, S. Biochemistry 2001, 40, 10326-10333). Another goal is to evaluate the sensitivity of the calculated ionization free energies and apparent pKa values on local structural fluctuations, which do not alter the structural convergence to a particular architecture, by using a complete ensemble of solution NMR structures and the NMR average minimized structure of FNfn10 (Main, A. L.; Harvey, T. S.; Baron, M.; Boyd, J.; Campbell, I. D. Cell 1992, 71, 671-678). Our calculations demonstrate that, at high ionic strength, FNfn10 is more stable at low pH compared to neutral pH, in overall agreement with experimental data. This behavior is attributed to contributions from unfavorable Coulombic interactions in a surface patch for the pairs Asp7-Glu9 and Asp7-Asp23. The unfavorable interactions are decreased at low pH, where the acidic residues become neutral, and are further decreased at high ionic strength because of increased screening by salt ions. Elimination of the unfavorable interactions in the theoretical mutants Asp7Asn (D7N) and Asp7Lys (D7K) produce higher calculated stabilities at neutral pH and any ionic strength compared to the wild-type, in agreement with the experimental data. We also discuss subtleties in the calculated apparent pKa values and ionization free energies, which are not in agreement with the experimental data. This work demonstrates that comparative electrostatic calculations can provide rapid predictions of pH-dependent properties of proteins and can be significant aids in guiding the design of proteins with tailored properties.     

Complete measurement of the pKa values of the carboxyl and imidazole groups in Bacillus circulans xylanase.

Electrostatic interactions in proteins can be dissected experimentally by determining the pKa values of their constituent ionizable amino acids. To complement previous studies of the glutamic acid and histidine residues in Bacillus circulans xylanase (BCX), we have used NMR methods to measure the pKa s of the seven aspartic acids and the C-terminus of this protein. The pKa s of these carboxyls are all less than the corresponding values observed with random coil polypeptides, indicating that their ionization contributes favorably to the stability of the folded enzyme. In general, the aspartic acids with the most reduced pKa s are those with limited exposure to the solvent and a high degree of conservation among homologous xylanases. Most dramatically, Asp 83 and Asp 101 have pKa s < 2 and thus remain deprotonated in native BCX under all conditions examined. Asp 83 is completely buried, forming a strong salt bridge with Arg 136. In contrast, Asp 101 is located on the surface of the protein, stabilized in the deprotonated form by an extensive network of hydrogen bonds involving an internal water molecule and the neutral side-chain and main-chain atoms of Ser 100 and Thr 145. These data provide a complete experimental database for theoretical studies of the ionization behavior of BCX under acidic conditions.     

Relationship between electrostatics and redox function in human thioredoxin: characterization of pH titration shifts using two-dimensional homo- and heteronuclear NMR.

The electrostatic behavior of potentially titrating groups in reduced human thioredoxin was investigated using two-dimensional (2D) 1H and 15N nuclear magnetic resonance (NMR) spectroscopy. A total of 241 chemical shift titration curves were measured over the pH range of 2.1-10.6 from homonuclear 1H-1H Hartmann-Hahn (HOHAHA) and heteronuclear 1H-15N Overbodenhausen correlation spectra. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the determination of pKas for certain isolated ionizable groups, including the single histidine residue at position 43 (pKa = 5.5 +/- 0.1) and a number of aspartic and glutamic acid carboxylate groups. Many of the titration curves demonstrate complex behavior due to the effects of interacting titrating groups, the long range of electrostatic interactions through the protein interior, and, perhaps, pH-induced conformational changes on the chemical shifts. Unambiguous assignment of the pKas for most of the 38 potentially ionizing groups of human thioredoxin could therefore not be made. In addition, there was no clear evidence that Asp-26 titrates in a manner corresponding to that observed in the Escherichia coli protein [Dyson, H. J., Tennant, L. L., & Holmgren, A. (1991) Biochemistry 30, 4262-4268]. The pKas of the active site cysteines were measured, however, with Cys-32 having an anomalously low value of 6.3 +/- 0.1 and that of Cys-35 between 7.5 and 8.6. These pKas are in agreement with proposed mechanisms for redox catalysis of thioredoxin and previously measured pKas within the active site of E. coli thioredoxin [Kallis, G. B., & Holmgren, A. (1980) J. Biol. Chem. 255, 10261-10265]. The stabilization of a thiolate anion at physiological pH can be explained by the interaction of the S gamma of Cys-32 with the amide of Cys-35 observed in the previously determined high-resolution solution structure of reduced human thioredoxin [Forman-Kay, J. D., Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2685-2698].     

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.     

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.     

pH-Dependent Conformational Changes in Proteins and Their Effect on Experimental pKas: The Case of Nitrophorin 4

The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis, substrate binding and protein structure. Due to interactions with the protein environment, their pK_as can be shifted from their solution values and, if a protein has two stable conformations, it is possible for a residue to have different “microscopic”, conformation-dependent pK_a values. In those cases, interpretation of experimental measurements of the pK_a is complicated by the coupling between pH, protonation state and protein conformation. We explored these issues using Nitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation where NO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open state from which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pK_as: 8.5 in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopic values to the experimentally observed “apparent” pK_a, obtaining a value of 6.5, in excellent agreement with experimental data. This value must be interpreted as the pH at which the closed to open population transition takes place. More generally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentally observed ensemble dependent apparent pK_as and that the insight gained in the relatively simple case of NP4 can be useful in several more complex cases involving a pH dependent transition, of great biochemical interest.     

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.     

Effect of pH on substrate and inhibitor kinetic constants of human liver alanine aminopeptidase. Evidence for two ionizable active center groups.

The presence of at least two ionizable active center groups has been detected by a study of the effect of pH upon catalysis of hydrolysis of L-alanyl-beta-naphthylamide by human liver alanine aminopeptidase and upon the inhibition of hydrolysis by inhibitors and substrate analogs. Octanoic acid, octylamine, and peptide inhibitors have been found to be competitive inhibitors and are therefore thought to bind the active center. L-Phe was previously shown to bind the active center since it was found to be a competitive inhibitor of the hydrolysis of tripeptide substrates (Garner, C. W., and Behal, F. J. (1975), Biochemistry 14, 3208). A plot of pKm vs. pH for the substrate L-Ala-beta-naphthylamide showed that binding decreased below pH 5.9 and above 7.5, the points at which the theoretical curve undergoes an integral change in slope. These points are interpreted as the pKa either of substrate ionizable groups or binding-dependent enzyme active center groups. Similar plots of pKm vs. pH for L-alanyl-p-nitroanilide (as substrate) and pKi vs. pH for L-Leu-L-Leu-L-Leu and D-Leu-L-Tyr (as inhibitors) gave pairs fo pKa values of 5.8 and 7.4, 6.0 and 7.5, and 5.7 and 7.5, respectively. All the above substrates (and D-Leu-L-Tyr) have pKa values near 7.5; therefore, the binding-dependent group with a pKa value near 7.5 is possibly this substrate group. Similar plots of pKi vs. pH for the inhibitors L-Phe, L-Met, L-Leu, octylamine, and octanoic acid had only one bending point at 7.7, 7.6, 7.4, 6.3, and 5.9, respectively. Amino acid inhibitors, octylamine, and octanoic acid have no groups with pKa values between 5 and 9. These data indicate that there are two active center ionizable groups with pKa values of approximately 6.0 and 7.5 which are involved in substrate binding or inhibitory amino acid binding but not in catalysis since Vmax was constant at all pH values tested.     

Increasing protein stability by altering long-range coulombic interactions.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.     

Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.     

A multinuclear NMR study of the active site of an endoglucanase from a strain of Bacillus. Use of Trp residues as structural probes.

In the hydrolytic reaction catalyzed by an endoglucanase from a Bacillus strain (endoglucanase K), 2 of 12 Trp residues, Trp174 and Trp243, are responsible for binding of the substrate and/or for the catalysis (Kawaminami, S., Ozaki, K., Sumitomo, N., Hayashi, Y., Ito, S., Shimada, I., and Arata, Y. (1994) J. Biol. Chem. 269, 28752-28756). Here we report results of a stable isotope-aided NMR analysis of the active site of endoglucanase K, using Trp174 and Trp243 as structural probes. Hydrogen-deuterium exchange experiments performed for the NH protons of main and side chains of Trp residues revealed that Trp174 and Trp243 are located in the hydrophilic and hydrophobic microenvironments in the active site, respectively. We also carried out pH titration experiments for indole C2 proton resonances of Trp residues and measured the pH dependence of specific activities for wild-type endoglucanase K and its mutants in which Glu or Asp residues are replaced with their respective amide forms. On the basis of the results obtained from the present study, we conclude that (a) Glu130 and Asp191, which are in spatial proximity to Trp174 and Trp243 in the active site, play a crucial role in the enzymatic activity; (b) Glu130 and Asp191 interact with each other in the active site, leading to an increase in the pKa values to 5.5 for both amino acid residues; and (c) the pKa values of Glu130 and Asp191 would lead to an unusually narrow pH-activity profile of the endoglucanase K.     

Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering

The dielectric constant in the active site cleft of subtilisin from Bacillus amyloliquefaciens has been probed by mutating charged residues on the rim and measuring the effect on the pKa value of the active site histidine (His64) by kinetics. Mutation of a negatively charged surface residue, which is 12 to 13 A from His64, to an uncharged one Asp----Ser99) lowers the pKa of the histidine by up to 0.4 unit at low ionic strength (0.005 to 0.01 M). This corresponds to an apparent dielectric constant of about 40 to 50 between Asp99 and His64. The mutation is in an external loop that is known to tolerate a serine at position 99 from homologies with subtilisins from other bacilli. The environment between His64 and Asp99 is predominantly protein. Another charged residue that is at a similar distance from His64 (14 to 15 A) and is also in an external loop that is known to tolerate a serine residue is Glu156, at the opposite side of the active site. There is only water in a direct line between His64 and Glu156. Mutation of Glu----Ser156 also lowers the pKa of His64 by up to 0.4 unit at low ionic strength. This change again corresponds to an apparent dielectric constant of about 40 to 50. The pKa values were determined from the pH dependence of kcat/KM for the hydrolysis of peptide substrates, with a precision of typically +/- 0.02 unit. The following suggests that the changes in pKa are real and not artefacts of experimental conditions: Hill plots of the data for pKa determination have gradients (h) of -1.00(+/- 0.02), showing that there are negligible systematic deviations from theoretical ionization curves involving a monobasic acid: the pH dependence for the hydrolysis of two different substrates (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl p-nitroanilide and benzoyl-L-valyl-L-glycyl-L-arginyl p-nitroanilide) gives identical results so that the pKa is independent of substrate; the pH dependence is unaffected by changing the concentration of enzyme, so that aggregation is not affecting the results; the shift in pKa is masked by high ionic strength, as expected qualitatively for ionic shielding of electrostatic interactions.     

In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core

The crystal structure of the staphylococcal nuclease mutant V66K, in which valine 66 is replaced by lysine, has been solved at 1.97 A resolution. Unlike lysine residues in previously reported protein structures, this residue appears to bury its side-chain in the hydrophobic core without salt bridging, hydrogen bonding or other forms of electrostatic stabilization. Solution studies of the free energy of denaturation, delta GH2O, show marked pH dependence and clearly indicate that the lysine residue must be deprotonated in the folded state. V66K is highly unstable at neutral pH but only modestly less stable than the wild-type protein at high pH. The pH dependence of stability for V66K, in combination with similar measurements for the wild-type protein, allowed determination of the pKa values of the lysine in both the denatured and native forms. The epsilon-amine of this residue has a pKa value in the denatured state of 10.2, but in the native state it must be 6.4 or lower. The epsilon-amine is thus deprotonated in the folded molecule. These values enabled an estimation of the epsilon-amine's relative change in free energy of solvation between solvent and the protein interior at 5.1 kcal/mol or greater. This implies that the value of the dielectric constant of the protein interior must be less than 12.8. Lysine is usually found with the methylene groups of its side-chain partly buried but is nevertheless considered a hydrophilic surface residue. It would appear that the high pKa value of lysine, which gives it a positive charge at physiological pH, is the primary reason for its almost exclusive confinement to the surface proteins. When deprotonated, this amino acid type can be fully incorporated into the hydrophobic core.     

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)     

Binding of calcium ions to bacteriorhodopsin.

Adding Ca2+ or other cations to deionized bacteriorhodopsin causes a blue to purple color shift, a result of deprotonation of Asp85. It has been proposed by different groups that the protonation state of Asp85 responds to the binding of Ca2+ either 1) directly at a specific site in the protein or 2) indirectly through the rise of the surface pH. We tested the idea of specific binding of Ca2+ and found that the surface pH, as determined from the ionization state of eosin covalently linked to engineered cysteine residues, rises about equally at both extracellular and cytoplasmic surfaces when only one Ca2+ is added. This precludes binding to a specific site and suggests that rather than decreasing the pKa of Asp85 by direct interaction, Ca2+ increases the surface pH by binding to anionic lipid groups. As Ca2+ is added the surface pH rises, but deprotonation of Asp85 occurs only when the surface pH approaches its pKa. The nonlinear relationship between Ca2+ binding and deprotonation of Asp85 from this effect is different in the wild-type protein and in various mutants and explains the observed complex and varied spectral titration curves.     

Role of protonatable groups of bovine heart bc(1) complex in ubiquinol binding and oxidation.

The pH dependence of the initial reaction rate catalyzed by the isolated bovine heart ubiquinol-cytochrome c reductase (bc1 complex) varying decylbenzoquinol (DBH) and decylbenzoquinone (DB) concentrations was determined. The affinity for DBH was increased threefold by the protonation of a group with pKa = 5.7 +/- 0.2, while the inhibition constant (Ki) for DB decreased 22 and 2.8 times when groups with pKa = 5.2 +/- 0.6 and 7.7 +/- 0.2, respectively, were protonated. This suggests stabilization of the protonated form of the acidic group by DBH binding. Initial rates were best fitted to a kinetic model involving three protonatable groups. The protonation of the pKa approximately 5.7 group blocked catalysis, indicating its role in proton transfer. The kinetic model assumed that the deprotonation of two groups (pKa values of 7.5 +/- 0.03 and approximately 9.2) decreases the catalytic rate by diminishing the redox potential of the iron-sulfur (Fe-S) cluster. The protonation of the pKa approximately 7.5 group also decreased the reaction rate by 80-86%, suggesting its role as acceptor of a proton from ubiquinol. The lack of effect on the Km for DBH when the pKa 7.5-7.7 group is deprotonated suggests that hydrogen bonding to this residue is not the main factor that determines substrate binding to the Qo site. The possible relationship of the pKa 5.2-5.7 and pKa 7.5-7.7 groups with Glu272 of cytochrome b and His161 of the Fe-S protein is discussed.