Individual pKa Values Research Articles

The Effect of pH on the Structure and Lateral Organization of Cardiolipin in Langmuir Monolayers.

Cardiolipin (CL) is a unique phospholipid featuring a dimeric structure. With its four alkyl chains, it has a large hydrophobic region and the charged hydrophilic head group is relatively small. Biological membranes exhibit CL exclusively in the inner bacterial and mitochondrial membranes. Alteration of CL packing can lead to structural changes and membrane instabilities. One environmental influence is the change in pH. Since the acidic properties of the phosphate head groups remain still controversial in literature, this work focusses on the influence of pH on the ionization degree of CL. For the analyses, surface pressure (π) - molecular area (A) isotherm experiments were combined with total reflection X-ray fluorescence (TRXF) and grazing incidence X-ray diffraction (GIXD). Continuous ionization with a high CL packing density was observed in the monolayer over a wide pH range. No individual pKa values can be assigned to the two phosphate groups, but mutual influence is observed.

Computing Surface Acidity Constants of Proton Hopping Groups from Density Functional Theory-Based Molecular Dynamics: Application to the SnO2(110)/H2O Interface.

Proton transfer at metal oxide/water interfaces plays an important role in electrochemistry, geochemistry, and environmental science. The key thermodynamic quantity to characterize this process is the surface acidity constant. An ab initio method that combines density functional theory-based molecular dynamics (DFTMD) and free energy perturbation theory has been established for computing surface acidity constants. However, it involves a reversible proton insertion procedure in which frequent proton hopping, e.g., for strong bases and some oxide surfaces (e.g., SnO2), can cause instability issues in electronic structure calculation. In the original implementation, harmonic restraining potentials are imposed on all O-H bonds (denoted by the VrH scheme) to prevent proton hopping and thus may not be applicable for systems involving spontaneous proton hopping. In this work, we introduce an improved restraining scheme with a repulsive potential Vrep to compute the surface acidities of systems in which proton hopping is spontaneous and fast. In this Vrep scheme, a Buckingham-type repulsive potential Vrep is applied between the deprotonation site and all other protons in DFTMD simulations. We first verify the Vrep scheme by calculating the pKa values of H2O and aqueous HS- solution (i.e., strong conjugate bases) and then apply it to the SnO2(110)/H2O interface. It is found that the Vrep scheme leads to a prediction of the point of zero charge (PZC) of 4.6, which agrees well with experiment. The intrinsic individual pKa values of the terminal five-coordinated Sn site (Sn5cOH2) and bridge oxygen site (Sn2ObrH+) are 4.4 and 4.7, respectively, both being almost the same as the PZC. The similarity of the two pKa values indicates that dissociation of terminal water has almost zero free energy at this proton hopping interface (i.e., partial water dissociation), as expected from the acid-base equilibrium on SnO2.

Individual pK a Values of Tobramycin, Kanamycin B, Amikacin, Sisomicin, and Netilmicin Determined by Multinuclear NMR Spectroscopy.

NMR spectroscopy is a powerful technique for separating and measuring each distinct pKa value of the amino groups around aminoglycoside antibiotics. Unambiguous assignments were made for each individual amine substituent on 2-deoxystreptamine, tobramycin, kanamycin B, amikacin, sisomicin, and netilmicin using variations in the NMR spectroscopic chemical shift (δ) with 1H, 13C, and 15N HMBC; the individual pKa values of netilmicin are reported for the first time.

A New Constant pH Method to Simultaneously Predict pH-Induced Conformational Changes and Individual PKA Values in Large Biomolecules

We describe a constant pH MD method that integrates a new FEP replica-exchange implementation for titratable residues into the Hamiltonian pH replica-exchange routine. Using the high performance OpenMM engine, during each FEP/REMD stage, a switch of protonation probabilities is attempted between 2 adjacent replicas (for a randomly chosen set of residues with a conventional Metropolis criterion). This FEP scheme enables a gradual evolution of the force field representation of protonation states for each residue independently, leading to a more precise calculation of protonation probabilities. Any net charge discrepancies arising from switches of protonation coupling parameters in systems with explicit solvent are addressed with an adaptive technique to preserve the overall zero charge for all replicas individually after each exchange step. Each replica runs on a single GPU, enabling linear scaling of N replicas on N GPU; this parallelization strategy, combined with advanced sampling, results in a significantly faster time-to-solution than other constant pH methods. Thus, the new method yields predictions of conformational states corresponding to given pH values with considerably reduced computational expense. Notably, the implementation's constant pH and MD modules are independent, rendering the method adaptable to a variety of propagation techniques and advanced sampling methods. The results obtained for small-, medium- and large-scale pH-dependent protein systems will be discussed to illustrate the performance of the current version of the method.

Amino acid-functionalized carbon nanotube framework as a biomimetic catalyst for cleavage of glycosidic bonds

In this work, carbon nanotubes (CNTs) functionalized by acidic amino acids were used as a framework, which aims to form a mimetic structure of an active site of the glycoside hydrolases. It was demonstrated that the glycosidic bonds of the disaccharides were cleaved by the fabricated biofunctionalized CNTs. It was implied that the number of carboxyl groups and their individual pKa values in the amino acids, and the distance between the NH2 and the side chain carboxyl groups of the amino acid are predominant factors for determining the reaction efficiency and the optimum pH. It was suggested that glutamic acid functionalized CNTs framework showed the highest efficiency in the cleavage of glycosidic bond of cellobiose than other acidic biomolecules. It was also suggested that the glutamic acid functionalized CNT framework showed preference to the types of glycosidic bonds in the following order: β-1,2-glycoside > β-1,4-glycoside > α-1,4-glycoside α-1,1-glycoside bond.

Synthesis, Structural Characterization, and Quantitative Basicity Studies of Lithium Zirconimidate Complexes

Synthesis, Structural Characterization, and Quantitative Basicity Studies of Lithium Zirconimidate Complexes

Increasing the Reactivity of an Artificial Dithiol−Disulfide Pair through Modification of the Electrostatic Milieu

The thiol-disulfide exchange reaction plays a central role in the formation of disulfide bonds in newly synthesized proteins and is involved in many aspects of cellular metabolism. Because the thiolate form of the cysteine residue is the key reactive species, its electrostatic milieu is thought to play a key role in determining the rates of thiol disulfide exchange reactions. While modest reactivity effects have previously been seen in peptide model studies, here, we show that introduction of positive charges can have dramatic effects on disulfide bond formation on a structurally restricted surface. We have studied properties of vicinal cysteine residues in proteins using a model system based on redox-sensitive yellow fluorescent protein (rxYFP). In this system, the formation of a disulfide bond between two cysteines Cys149 and Cys202 is accompanied by a 2.2-fold decrease in fluorescence. Introduction of positively charged amino acids in the proximity of the two cysteines resulted in an up to 13-fold increase in reactivity toward glutathione disulfide. Determination of the individual pK(a) values of the cysteines showed that the observed increase in reactivity was caused by a decrease in the pK(a) value of Cys149, as well as favorable electrostatic interactions with the negatively charged reagents. The results presented here show that the electrostatic milieu of cysteine thiols in proteins can have substantial effects on the rates of the thiol-disulfide exchange reactions.

Theoretical calculations of acid-dissociation constants of proteins

The purpose of this review is to introduce several computational procedures for the determination of acid-dissociation constants (pKa) of titratable groups in proteins. Several concepts, such as continuum electrostatics and the exact meaning of intrinsic and apparent pKas, will be explained in some detail. Each of the methods will be judged on its merits, and some comparisons between the methods will be presented. While the emphasis of this review will be on theoretical formulations, the experimental determination by means of nuclear magnetic resonance will be briefly explained. The determination of individual pKa values by nuclear magnetic resonance in combination with computationally determined pKas can provide unique information about the pH-dependent properties of proteins and their complexes with peptides, DNA, and ligands.Key words: acid-dissociation constants, NMR, continuum electrostatics, dielectric constant of proteins, Monte Carlo, molecular dynamics.

9] Calculations of proton-binding thermodynamics in proteins

Computational models of proton binding can range from the chemically complex and statistically simple (as in the quantum calculations) to the chemically simple and statistically complex. Much progress has been made in the multiple-site titration problem. Calculations have improved with the inclusion of more flexibility in regard to both the geometry of the proton binding and the larger scale protein motions associated with titration. This article concentrated on the principles of current calculations, but did not attempt to survey their quantitative performance. This is (1) because such comparisons are given in the cited papers and (2) because continued developments in understanding conformational flexibility and interaction energies will be needed to develop robust methods with strong predictive power. Nevertheless, the advances achieved over the past few years should not be underestimated: serious calculations of protonation behavior and its coupling to conformational change can now be confidently pursued against a backdrop of increasing understanding of the strengths and limitations of such models. It is hoped that such theoretical advances will also spur renewed experimental interest in measuring both overall titration curves and individual pKa values or pKa shifts. Exploration of the shapes of individual titration curves (as measured by Hill coefficients and other parameters) would also be useful in assessing the accuracy of computations and in drawing connections to functional behavior.

Theoretical calculations of acid-dissociation constants of proteins.

The purpose of this review is to introduce several computational procedures for the determination of acid-dissociation constants (pKa) of titratable groups in proteins. Several concepts, such as continuum electrostatics and the exact meaning of intrinsic and apparent pKas, will be explained in some detail. Each of the methods will be judged on its merits, and some comparisons between the methods will be presented. While the emphasis of this review will be on theoretical formulations, the experimental determination by means of nuclear magnetic resonance will be briefly explained. The determination of individual pKa values by nuclear magnetic resonance in combination with computationally determined pKas can provide unique information about the pH-dependent properties of proteins and their complexes with peptides, DNA, and ligands.

Measurement of the individual pKa values of acidic residues of hen and turkey lysozymes by two-dimensional 1H NMR

The pH dependence of the two-dimensional 1H nuclear magnetic resonance spectra of hen and turkey egg-white lysozymes has been recorded over the pH range 1-7. By monitoring the chemical shifts of the resonances of the various protons of ionizable residues, individual pKa values for the acidic residues have been determined for both proteins. The pKa values are displaced, with the exception of those of the residues in the active site cleft, by an average of 1 unit to low pH compared to model compounds.

Determination of the side chain pKa values of the lysine residues in calmodulin.

The 7 Lys residues in mammalian calmodulin (CaM) were reductively methylated with 13C-enriched formaldehyde and studied by (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR. The apo- and Ca(2+)-forms of CaM, as well as a complex with a 22-residue peptide which comprises the CaM binding region of myosin light chain kinase were studied. The complete assignment of the two-dimensional NMR spectra was obtained by site-directed mutagenesis (Lys-->Gln) of all the Lys. The pKa values for the individual Lys could be determined by pH titration experiments. In Ca(2+)-CaM, the pKa values range from 9.29 (Lys-75) to 10.23 (Lys-77). The Lys in apo-CaM have higher pKa values than those in Ca(2+)-CaM. The binding of the myosin light chain kinase peptide gives rise to an increase of the pKa values of Lys-148 and Lys-75 by 0.5 and 0.8 pH units, respectively; this results from the relocation of their side chains to a completely solvent accessible state. The changes in the pKa values upon binding Ca2+ or the myosin light chain kinase peptide show a remarkable correlation with earlier reported chemical reactivity changes. Thus, our results indicate that pKa values, rather than structural and steric effects, play the dominant role in determining the reactivity of Lys side chains towards small electrophilic chemical modification reagents. The methodology used here could prove useful for the determination of individual pKa values in other proteins.

1H-n.m.r. study of the solution properties and secondary structure of neurotoxin III from the sea anemone Anemonia sulcata.

The solution properties, secondary structure and global fold of the 27-residue polypeptide neurotoxin III (ATX III), from the sea anemone Anemonia sulcata, have been investigated using high-resolution 1H-n.m.r. spectroscopy. Studies of the concentration dependence of the n.m.r. spectrum indicate that the molecule self-associates in the millimolar concentration range useable for n.m.r. analysis, the association being less pronounced at acidic pH values. The dependence on pH of association implies that electrostatic interactions play a role in this process, while the significant concentration-dependent shifts of the aromatic resonances of Tyr-7 and Trp-13 indicate that hydrophobic interactions also contribute. Individual pKa values have been determined for most ionizable groups in the molecule. Sequence-specific resonance assignments were obtained for all protons using a range of two-dimensional homonuclear-correlated and nuclear-Overhauser-effect (nOe) spectra. The secondary structure of the polypeptide was identified from sequential (i, i+1) and medium-range (i, i+2/3/4) nOe connectivities, NH to C alpha H coupling constants, C alpha H chemical shifts, and the location of slowly exchanging backbone-amide protons. ATX III contains no regular alpha-helix or beta-sheet, consisting instead of a network of reverse turns. nOe connectivities between half-cystine residues are consistent with the disulphide pairings 3-17, 4-11 and 6-22. ATX III has a well-defined structure and appears to lack the disordered loop which, in the longer sea anemone toxins (46-49 residues), may be part of the receptor-binding surface.

Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.

In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.

Proton NMR study of the solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus

The solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I) have been investigated by high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz. The pH dependence of the spectra has been examined over the range 1.1-12.2 at 27 degrees C. Individual pKa values have been obtained for the alpha-ammonium group of Ala-1 (8.6) and the side chains of Glu-8 (3.7), Tyr-36 (10.9), and Tyr-37 (10.8). For the remaining seven carboxyl groups in the molecule (from five Asp, Glu-31, and the C-terminus), four pKa values, viz., 2.8, 3.5, 4.1 and 6.4, can be clearly identified. The five Lys residues titrate in the range 10.5-11, but individual pKa values could not be obtained because of peak overlap. Conformational changes associated with the protonation of carboxylates occur below pH 4, while in the alkaline pH range major unfolding occurs above pH 10. The molecule also unfolds at elevated temperatures, having a transition temperature of ca. 55 degrees C at pH 5.25. Exchange of the backbone amide protons has been monitored at various values of pH and temperature in the ranges pH 4-5 and 12-27 degrees C. Up to 18 slowly exchanging amides are observed, consistent with the existence of a core of hydrogen-bonded secondary structure, most probably beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)

A computer method for the spectrophotometric determination of overlapping pKa values

A least-squares computer method has been developed to obtain two pKa values from spectrophotometric data in instances when the values are so close together that the calculations usually applied for individual pKa values are not appropriate. A scaling procedure improves the condition of the matrix that is constructed in solving the sets of simultaneous equations. Two examples of applications of the method are given.