1H nuclear magnetic resonance studies of histidine-containing di- and tripeptides. Estimation of the effects of charged groups on the pKa value of the imidazole ring.

The nmr titration curves of chemical shifts versus pH were observed for the protons of various histidine-containing di- and tripeptides. With these results, the macroscopic pK_a values and the chemical shifts intrinsic to each ionic species were determined by a computer curve-fitting based on a simple acid dissociation sequence. The pK_a value of the imidazole ring in N-acetyl-L -histidine methylamide was assumed to represent the intrinsic (or unperturbed) pK_a of the imidazole rings of histidine having peptide linkages at both the CO and NH sides. The pK_a values of the imidazole rings observed for most di- and tripeptides were reasonably reproduced by simple calculations using the intrinsic value and the perturbations due to the CO₂^? and NH₃⁺ groups located at various positions. Some other factors affecting the pK_a value of the imidazole ring are also discussed.     

1H-nmr parameters of the common amino acid residues measured in aqueous solutions of the linear tetrapeptides H-Gly-Gly-X-L-Ala-OH

The ¹H-nmr chemical shifts and the spin–spin coupling constants of the common amino acid residues were measured in solutions of the linear tetrapeptides H-Gly-Gly-X-L -Ala-OH in D₂O and H₂O, the influence of X on the nmr parameters of the neighboring residues Gly 2 and Ala 4 was investigated. The titration parameters for the side chains of Asp, Glu, Lys, Tyr, and His were determined. The pK_a values obtained in D₂O, with the use of pH-meter readings with a combination glass electrode uncorrected for istope effects, were 0.06 pH units higher in the acidic range and 0.10 pH units higher in the basic range than the corresponding pK_a values in H₂O. This suggests that the present data are suitable “random-coil” ¹H-nmr parameters for conformational studies of polypeptide chains in D₂O and H₂O solutions.     

Influence of p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> Shifts on the Calculated Dipole Moments of Proteins

The protein dipole moment is a low-resolution parameter that characterizes the second-order charge organization of a biomolecule. Theoretical approaches to calculate protein dipole moments rely on pK _a values, which are either computed individually for each ionizable residue or obtained from model compounds. The influence of pK _a shifts are evaluated first by comparing calculated and measured dipole moments of β-lactoglobulin. Second, calculations are made on a dataset of 66 proteins from the Protein Data Bank, and average differences are determined between dipole moments calculated with model pK _as, pK _as derived using a Poisson–Boltzmann approach, and empirically-calculated pK _as. Dipole moment predictions that neglect pK _a shifts are consistently larger than predictions in which they are included. The importance of pK _a shifts are observed to vary with protein size, internal permittivity, and solution pH.     

The First and Second Dissociation Constants of Subtilisin BPN′-Plasminostreptin Complex Determined by a Polarographic Method,and the Effects of pH on Them

A polarographic method based on the Brdi?ka current (the polarographic catalytic hydrogen evolution current produced by proteins in the presence of cobalt salts) was applied to direct titration of subtilisin BPN′ (S.BPIST) with plasminostreptin (PS) at a concentration level of 10”⁸ m. The first and second dissociation constants of the S.BPN-PS complex were determined by fitting theoretical curves to the titration data, in which the multiple equilibria involving microscopically distinct forms of S.BPN-PS complex were taken into account. The intrinsic free energy change in the first binding of S.BPN′ to dimeric PS was larger than that in the second binding. The dependence of the microscopic dissociation constants of S.BPN′-PS complex on pH indicates the participation of ionizable groups of pK_a 8.0 and 9.4 in the complex formation. The repulsive effect between negatively charged molecules of S.BPN′ and PS in the complex formation at elevated pH is suggested.     

Theoretical studies on the redox-Bohr effect in cytochrome c 3 from Desulfovibrio vulgaris Hildenborough

 The pH dependence of the redox potentials in the tetrahemic cytochrome c ₃ from Desulfovibrio vulgaris Hildenborough (redox-Bohr effect) is here investigated using continuum electrostatics methods. The redox-Bohr effect seems to be associated with changes in the protonation state of charged residues in the protein, but the exact residues had not been identified. The global pK _a of this phenomenon is dependent on the redox state of the molecule, and the influence of the pH on the microscopic potential of each heme has been experimentally quantified. The availability of detailed experimental data provides us with important and unique guides to the performance of ab initio pK _a calculations aiming at the identification of the groups involved. These calculations were performed in several redox states along the reduction pathway, with the double objective of finding groups with redox-linked pK _a shifts, and absolute pK _as compatible with the redox-Bohr effect. The group with the largest pK _a shift along the reduction pathway is propionate D from heme I. Its effect on the redox potential of individual hemes, as calculated by electrostatic calculations, correlates very well with the experimental order of influence, making it a likely candidate. Abnormal titration of the same propionate has been experimentally observed on a homologous cytochrome c ₃ from a different strain, thus strengthening the theoretical result. However, its absolute calculated pK _a in the fully oxidised cytochrome is outside the zone where the phenomenon is known to occur, but the calculation shows a strong dependence on small conformational changes, suggesting large uncertainties in the calculated value. A group with a pK _a value within the experimentally observed range is propionate D from heme IV. Its influence on the redox potential of the hemes does not correlate with the experimental order, indicating that, although it may be one of the possible players on the phenomenon, it cannot be solely responsible for it. Mutation of the Lys45 residue is suggested as an indirect way of probing the importance of the propionate D from heme I in the mechanism. Non-heme groups may also be involved in this process; our calculations indicate His67 and the N-terminal as groups that may play a role. Accuracy and applicability of current continuum electrostatic methods are discussed in the context of this system. Received: 27 March 1997 / Accepted: 19 August 1997     

Nitrilotris(methylenephosphonates) in aqueous solution and solid state - dilatometric, potentiometric and NMR investigations

Dissociation and alkali complex formation equilibria of nitrilotris(methylenephosphonic acid) (NTMP, H₆L) have been studied by dilatometric, potentiometric and ³¹P NMR-controlled titrations. Dilatometry indicated the formation of alkali complexes ML (M=Li, Na, K, Rb, Cs) at high pH with a stability decreasing from Li to Cs. An efficient combination of potentiometric and NMR methods confirmed two types of alkali metal complexes MHL and ML. Stability constants for the equilibria following M⁺ + HL⁵⁻ ? MHL⁴⁻ and M⁺ + L⁶⁻ ? ML⁵⁻, respectively, were determined: logK_NaHL=1.08(0.07), logK_KHL=0.86(0.08), logK_NaL=2.24(0.03). Systematic errors are introduced by using alkali metal hydroxides as titrants for routine potentiometric determinations of dissociation constants pK_a5^app and pK_a6^app. Correction formulae were derived to convert actual dissociation constants pK_a into apparent dissociation constants pK_a^app (or vice versa). The actual dissociation constants were found: pK_a5(H₂L⁴⁻ ? H⁺ + HL⁵⁻)=7.47(0.03) and pK_a6(HL⁵⁻ ? H⁺ + L⁶⁻)=14.1(0.1). The anisotropy of ³¹P chemical shifts of salts M_nH_6 − nL (M=Li, Na, n=0-5) is more sensitive towards titration (n) than isotropic solution state chemical shifts.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues: II. Microenvironments of histidine-12 and histidine-119 of bovine pancreatic ribonuclease a

The NMR titration curves of proton chemical shifts were observed for the C₂ protons of histidine residues in intact bovine pancreatic RNAase A (EC 3.1.27.5) and carboxyalkylated RNAase A. By comparing the methyl region of NMR spectra, the 250–340 nm region of circular dichfoic spectra, and the NMR titration curves of tyrosine ring protons among intact and modified RNAase A, it was ascertained that the carboxyalkylation of histidine residues at position 12 or 119 did not make any appreciable conformational changes to RNAase A. With the pK values determined for intact and modified RNAase A, the microscopic pK values and molar ratios of tautomers were estimated for His-12 and His-119 by means of the procedure described in the preceding paper. The estimated microscopic pK values of tautomers were 6.2 for the N₁-H tautomer of His-12, more than 8 for the N₃-H tautomer of His-12, 7.0 for the N₁-H tautomer of His-119, and 6.4 for the N₃-Hf tautomer of His-119, respectively. These values were interpreted in terms of the microscopic environments surrounding the histidine residues. The microscopic structure estimated in the present study was discussed, comparing it with those from X-ray crystallography and hydrogen-tritium (or hydrogen-deuterium) exchange technique.     

Characterization and determination of titratable groups of proteins by linearization of titration curves. II. Application to lysozyme

The potentiometric acid-base titration curve of fully protonated lysozyme at ionic strengths of 0.10 and 1.0 m has been performed. The stoichiometry and the pK_a values of each titratable group have been determined through the linearization of titration curves. Two types of carboxylic groups with pK_a values of 3.76 and 5.02, the imidazole group with pK_a 7.37 and the amine group with pK_a 9.63, have been identified at an ionic strength of 0.10 m at 25.0°C. The number of titratable groups found per mole of protein has been 5.12 and 5.60 for the two types of carboxylic groups, 1.13 for the imidazole group, and 3.19 for the amino groups. The endpoint of the titration of the protein obtained by this method accords quite well with the endpoint obtained by the use of Gran function applied to the excess of strong base.     

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.     

Protonation of kanamycin A: Detailing of thermodynamics and protonation sites assignment

Protonation of an aminoglycoside antibiotic kanamycin A sulfate was studied by potentiometric titrations at variable ionic strength, sulfate concentration and temperature. From these results the association constants of differently protonated forms of kanamycin A with sulfate and enthalpy changes for protonation of each amino group were determined. The protonation of all amino groups of kanamycin A is exothermic, but the protonation enthalpy does not correlate with basicity as in a case of simple polyamines. The sites of stepwise protonation of kanamycin A have been assigned by analysis of ¹H-¹³C-HSQC spectra at variable pH in D₂O. Plots of chemical shifts for each H and C atom of kanamycin A vs. pH were fitted to the theoretical equation relating them to pK_a values of ionogenic groups and it was observed that changes in chemical shifts of all atoms in ring C were controlled by ionization of a single amino group with pK_a 7.98, in ring B by ionization of two amino groups with pK_a 6.61 and 8.54, but in ring A all atoms felt ionization of one group with pK_a 9.19 and some atoms felt ionization of a second group with pK_a 6.51, which therefore should belong to amino group at C3 in ring B positioned closer to the ring A while higher pK_a 8.54 can be assigned to the group at C1. This resolves the previously existed uncertainty in assignment of protonation sites in rings B and C.     

Proton NMR investigation of heme and surrounding proton in low-spin cyanide-ligated bacterial hemoglobin from Vitreoscilla

¹H NMR spectra of low-spin cyanide-ligated bacterial hemoglobin fromVitreoscilla (VtHb-CN) are reported. The assignments of the¹H NMR spectra of VtHb-CN have been made through MCOSY, NOESY, 1D TOE and SUPERWEFT experiments. Almost all resonance peaks of heme and ligated His85 are identified. The spin-lattice relaxation timeT ¹’s and the variation relationships of chemical shifts of these peaks with temperature have been acquired, from which the distances between the measured protons and Fe³⁺, and the diamagnetic chemical shifts have been acquired, respectively. The ionization constants of pK _a’s of ligated His85 are determined through pH titration of chemical shift, which is 4.95 for ligated His85 C₂H proton. The lower pK _a is attributed to the influence of the Fe³⁺ of carrying positive charge and the coordination of His85 and Fe³⁺ of heme.     

pH-dependent redox and CO binding properties of chelated protoheme-<Emphasis Type="SmallCaps">l</Emphasis>-histidine and protoheme-glycyl-<Emphasis Type="SmallCaps">l</Emphasis>-histidine complexes

The pH dependence of redox properties, spectroscopic features and CO binding kinetics for the chelated protohemin-6(7)-l-histidine methyl ester (heme-H) and the chelated protohemin-6(7)-glycyl-l-histidine methyl ester (heme-GH) systems has been investigated between pH 2.0 and 12.0. The two heme systems appear to be modulated by four protonating groups, tentatively identified as coordinated H₂O, one of heme’s propionates, Nε of the coordinating imidazole, and the carboxylate of the histidine residue upon hydrolysis of the methyl ester group (in acid medium). The pK _a values are different for the two hemes, thus reflecting structural differences. In particular, the different strain at the Fe–N _ε bond, related to the different length of the coordinating arm, results in a dramatic alteration of the bond strength, which is much smaller in heme-H than in heme-GH. It leads to a variation in the variation of the pK a for the protonation of the N _ε of the axial imidazole as well as in the proton-linked behavior of the other protonating groups, envisaging a cross-talk communication mechanism among different groups of the heme, which can be operative and relevant also in the presence of the protein matrix. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

Reassignment of the active site histidines in ribonuclease A by selective deuteration studies.

The C₂H resonance of the active site histidine residue designated AS-2, which has the lower pK_a of the two active site histidines, has been correlated in both RNase A and RNase S by comparing the pH 3 to 5.5 regions of the chemical shift titration curves, the effect of the inhibitor CMP-3′ on the chemical shifts at pH 4.0, and the effect of Cu II on the line widths at pH 3.6. It has been demonstrated that resonance AS-2 is absent in the spectrum of RNase S′ reconstituted using S-peptide deuterated at the C₂ of His 12, and in that of the RNase S′-CMP-3′ complex. We thus demonstrate that histidine AS-2 is in fact His 12 in both enzymes. This finding is in agreement with out previous assignment of the exchangeable NH proton in RNase A to His 12, but reverses the assignments of the active site histidine C₂H resonances made earlier by other authors.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c in 0.15M KCl at 20°C have been obtained by timed titration (0.125–0.500 μmol/sec) from the isoionic points (pH 10.2–10.4) to pH 3 and back to the isoionic point. Computer-assisted (PROPHET) data acquisition and blank corrections give curves with good precision with a maximum standard deviation of 0.3 groups for an average error of 1%. The potentiometric titration curve of reduced cytochrome c is reversible within the precision of the method and for the pH range studied. The potentiometric curves for oxidized cytochrome c titrated upscale (pH 3–10) and downscale (pH 10–3) are not reversible. However, they show the same ionization behavior after the initial downscale titration. This is probably the result of a conformational change. Comparison of the data herein reported with the titration curves of oxidized cytochrome c already published by others indicates good agreement on the basis of a normalization of the concentration of protein or on the basis of 25 titrable groups between the acid end point and the isoionic pH. Titration of the 2 μmol imidazole in the upscale or downscale direction gives the correct analytical concentration and pK′ after correction for the solvent titration. Titration of reduced cytochrome c in the presence and absence of an additional equivalent of imidazole gave a difference titration curve, which indicates that a group on the protein shifts from pK′ 5.8 to pK′ 5.3 in the presence of imidazole. The pK′ of imidazole, in the presence of the protein, remains at a nearly normal value of 7.34.     

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.     

Practical acid dissociation constants,temperature coefficients,and buffer capacities for some biological buffers in solutions containing dimethyl sulfoxide between 25 and −12 °C

Acid dissociation constants (K_a), usually expressed as pK_a (-logK_a) can be considered as indices of acid-base equilibria in solution and their evaluation under the solution conditions that exist during the exposure of biological systems to low temperatures are as important as the measurement of pH per se. The assignment of pH1 standards to define pH1 scales in the binary mixed solvent, dimethyl sulfoxide-water (27), has provided the basis for measuring the pK_a1 values of some biological buffers in mixtures of Me₂SO and H₂O which have particular relevance to studies which demonstrate the “pH-dependent” recovery of smooth muscle after low-temperature storage (9, 31). “Practical” ionization conslants in water (pK′_a) and in 20% ($\text{w}\text{w}$) and 30% ($\text{w}\text{w}$) dimethylsulfoxide-water (pK_a1) have been measured by potentiometric titration of a range of zwitterionic buffer compounds at 25, 0, ?5.5, and ?12 °C together with the respective buffer capacities and temperature coefficients. Measurements have been made with reference to the relevant standard states for each solvent system, thereby endowing the values with as much thermodynamic significance as possible.     

Titration_DB: Storage and analysis of NMR‐monitored protein pH titration curves

NMR‐monitored pH titration experiments are routinely used to measure site‐specific protein pKa values. Accurate experimental pKa values are essential in dissecting enzyme catalysis, in studying the pH‐dependence of protein stability and ligand binding, in benchmarking pKa prediction algorithms, and ultimately in understanding electrostatic effects in proteins. However, due to the complex ways in which pH‐dependent electrostatic and structural changes manifest themselves in NMR spectra, reported apparent pKa values are often dependent on the way that NMR pH‐titration curves are analyzed. It is therefore important to retain the raw NMR spectroscopic data to allow for documentation and possible re‐interpretation. We have constructed a database of primary NMR pH‐titration data, which is accessible via a web interface. Here, we report statistics of the database contents and analyze the data with a global perspective to provide guidelines on best practice for fitting NMR titration curves. Titration_DB is available at http://enzyme.ucd.ie/Titration_DB . Proteins 2010. © 2009 Wiley‐Liss, Inc.