A simple chemical example of hierarchical thermodynamic interactions: the protonation equilibria of inorganic polyprotic acids

A general method for formulating complex thermodynamic systems in terms of hierarchical interactions has been developed, and has been applied in a previous analysis to hemoglobin oxygen binding data. Polyprotic acids can be considered a simple chemical model of thermodynamic interaction between ligand binding events. To further illustrate the hierarchical interaction approach it is applied to the analysis of the thermodynamic interactions between proton binding events in inorganic polyprotic acids. pK values for arsenate, carbonate, chromate, phosphate, phosphite, selenite, sulfide and sulfite were recast into hierarchical interaction terms. The intrinsic K(d,h) for protonation ranged from 8.8 x 10(-13) (M) for phosphate to 1.3 x 10(-6) (M) for chromate. Pairwise interactions (K(d,hh)) between protonation events ranged from 1.3 x 10(4) for phosphite to 9.4 x 10(5) for carbonate. Third order interactions (K(d,hhh)) were 0.91 and 0.51 for arsenate and phosphate, respectively, values relatively close to the no interaction value of 1. A principle feature of systems described by hierarchical interactions is that higher order interactions, representing more complex interactions, are less likely to be significant than lower order interactions, and this is further illustrated by these observations from polyprotic acids. The set of significant hierarchical interaction values can be used to predict values for as yet unobserved events, and projected pK values are made for all the polyprotic acids included in this study. Finally, application of this method to the protonation equilibria of water demonstrates a profound pairwise interaction between protonation events (K(d,hh) = 1.3 x 10(17)), which is attributed to oxygen's small size and lack of polarizability.     

Protonation free energy levels in complex molecular systems

All proteins, nucleic acids, and other biomolecules contain residues capable of exchanging protons with their environment. These proton transfer phenomena lead to pH sensitivity of many molecular processes underlying biological phenomena. In the course of biological evolution, Nature has invented some mechanisms to use pH gradients to regulate biomolecular processes inside cells or in interstitial fluids. Therefore, an ability to model protonation equilibria in molecular systems accurately would be of enormous value for our understanding of biological processes and for possible rational influence on them, like in developing pH dependent drugs to treat particular diseases. This work presents a derivation, by thermodynamic and statistical mechanical methods, of an expression for the free energy of a complex molecular system at arbitrary ionization state of its titratable residues. This constitutes one of the elements of modeling protonation equilibria. Starting from a consideration of a simple acid-base equilibrium of a model compound with a single tritratable group, we arrive at an expression which is of general validity for complex systems. The only approximation used in this derivation is the postulating that the interaction energy between any pair of titratable sites does not depend on the protonation states of all the remaining ionizable groups.     

Effect of protonation of nucleic acid bases on the energy of formation of the Watson-Crick pairs

Neutral and protonated nucleic bases and their complexes were calculated using a modified MNDO method. On the basis of the obtained proton affinities we conclude that proton transfer from positively charged amino acid residues to nucleic bases is quite possible. The protonation influence upon the structure and the energy of complementary base pairs was studied. The protonation of guanine is shown to stabilize the GC complex, but the protonation of cytosine destabilizes it. The energy of the AU pair increases upon protonation of adenine due to ion--dipole interactions. The protonation of uracil leads to a proton transfer between the bases and to the stabilization of the AU pair.     

Glutamate 268 regulates transport probability of the anion/proton exchanger ClC-5

The Cl(-)/H(+) exchange mediated by ClC transporters can be uncoupled by external SCN(-) and mutations of the proton glutamate, a conserved residue at the internal side of the protein. We show here for the mammalian ClC transporter ClC-5 that acidic internal pH led to a greater increase in currents upon exchanging extracellular Cl(-) for SCN(-). However, transport uncoupling, unitary current amplitudes, and the voltage dependence of the depolarization-induced activation were not altered by low pH values. Therefore, it is likely that an additional gating process regulates ClC-5 transport. Higher internal [H(+)] and the proton glutamate mutant E268H altered the ratio between ClC-5 transport and nonlinear capacitance, indicating that the gating charge movements in ClC-5 arise from incomplete transport cycles and that internal protons increase the transport probability of ClC-5. This was substantiated by site-directed sulfhydryl modification of the proton glutamate mutant E268C. The mutation exhibited small transport currents together with prominent gating charge movements. The charge restoration using a negatively charged sulfhydryl reagent reinstated also the WT phenotype. Neutralization of the charge of the gating glutamate 211 by the E211C mutation abolished the effect of internal protons, showing that the increased transport probability of ClC-5 results from protonation of this residue. S168P (a mutation that decreases the anion affinity of the central binding site) reduced also the internal pH dependence of ClC-5. These results support the idea that protonation of the gating glutamate 211 at the central anion-binding site of ClC-5 is mediated by the proton glutamate 268.     

Computational investigation of proton transfer,pKa shifts and pH‐optimum of protein–DNA and protein–RNA complexes

Protein–nucleic acid interactions play a crucial role in many biological processes. This work investigates the changes of pKa values and protonation states of ionizable groups (including nucleic acid bases) that may occur at protein–nucleic acid binding. Taking advantage of the recently developed pKa calculation tool DelphiPka, we utilize the large protein–nucleic acid interaction database (NPIDB database) to model pKa shifts caused by binding. It has been found that the protein's interfacial basic residues experience favorable electrostatic interactions while the protein acidic residues undergo proton uptake to reduce the energy cost upon the binding. This is in contrast with observations made for protein–protein complexes. In terms of DNA/RNA, both base groups and phosphate groups of nucleotides are found to participate in binding. Some DNA/RNA bases undergo pKa shifts at complex formation, with the binding process tending to suppress charged states of nucleic acid bases. In addition, a weak correlation is found between the pH‐optimum of protein–DNA/RNA binding free energy and the pH‐optimum of protein folding free energy. Overall, the pH‐dependence of protein–nucleic acid binding is not predicted to be as significant as that of protein–protein association. Proteins 2017; 85:282–295. © 2016 Wiley Periodicals, Inc.     

A theoretical study oh the effect of "bound" water on the proton chemical shifts of the nucleic acid bases.

Computations are performed on the proton chemical shifts due to hydrogen bonding between the purine and pyrimidine bases of the nucleic acids and water molecules of their first hydration shell. The water molecules should produce measurable shifts essentially for protons of the bases located close to the site of interaction. For the imino protons of the bases G-N1H and U-N3H participating in hydrogen bonding, the calculated delta delta is larger for the interaction of a base with a complementary base than for its interaction with water. Base pairing will thus produce a downfield shift in water but the measured delta delta due to pairing in this solvent will be smaller than in an inert solvent. Also, the chemical shift difference between G-N1H and U-N3H in water will be larger if the molecules are engaged in pairs than if they are not.     

The meaning of the genetic code: reconstruction of the stage of prebiologic evolution

According to a certain order in sets of the two first codon bases, 20 common amino acids can be divided into 5 families each containing 4 amino acids; the corresponding order in the distribution of codon bases can be easily detected, if common amino acids are distributed for the numbers of hydrogen atoms per molecule (Sukhodolets, 1980). In the present paper, the order in the distribution of codon bases is explained on the basis of the hypothesis claiming the prebiological existence of crystalline associates composed of amino acids and bases as free molecules. In these heterogeneous crystalline associates amino acids were analogs to the base douplets and the arrangement of molecules followed a certain rule, namely: 40 protons per molecular complex forming a standard structural compartment. It is proposed that the crystalline associates existed as lyotropic liquid crystals with hydrocarbons as solvent. The genetical code allows to discover two different original crystallization types for bases and amino acids. Therefore, the life possibly originates from combining in the same structure different crystallization patterns, which resulted in formation of a finite crystalline associate.     

Complexes of amino acids with a crown-ether derivative of 4-styrylpyridine. Monotopic or ditopic?

An E-4-styrylpyridine derivative endowed with 18-crown-6 as a substituent (E-1) was prepared and evaluated in acetonitrile as a potential ditopic ligand for protonated amino acids. The interactions of E-1 with the protonated amino acid perchlorates, ClO(4)(-) H(3)N(+)(CH(2))(n)COOH (n = 2, 5 and 10, A2, A5 and A10, respectively), were studied by optical methods, (1)H NMR and mass spectroscopy. Complex formation involves coordination of the ammonium ion at the crown ether moiety of E-1. The spectral changes were evaluated by comparison with results obtained on protonation of E-1 with HClO(4) and on association with ammonium perchlorate. Protonation by the protonated amino acid perchlorates was thwarted due to reversal of carboxyl/pyridinium pK(A) order in acetonitrile relative to water. Evidence for ditopic hydrogen bonding complex formation was especially sought for A10 because its CH(2) chain is sufficiently long to bridge the distance between the crown ether and pyridyl N sites of E-1. Despite some subtle hints to the contrary, the absence of NOE interaction between the pyridyl protons of E-1 and the methylene protons of A10 indicates that the E-1·A10 complex is in the main monotopic, as is the case for A2 and A5. The photophysical and photochemical behaviour of the complexes change significantly on protonation by HClO(4). The optical response of E-1 on binding the amino acids as ammonium salts allows convenient monitoring of complex formation.     

Interplay between local protein interactions and water bridging of a proton antenna carboxylate cluster

Proteins that bind protons at cell membrane interfaces often expose to the bulk clusters of carboxylate and histidine sidechains that capture protons transiently and, in proton transporters, deliver protons to an internal site. The protonation-coupled dynamics of bulk-exposed carboxylate clusters, also known as proton antennas, is poorly described. An essential open question is how water-mediated bridges between sidechains of the cluster respond to protonation change and facilitate transient proton storage. To address this question, here I studied the protonation-coupled dynamics at the proton-binding antenna of PsbO, a small extrinsinc subunit of the photosystem II complex, with atomistic molecular dynamics simulations and systematic graph-based analyses of dynamic protein and protein-water hydrogen-bond networks. The protonation of specific carboxylate groups is found to impact the dynamics of their local protein-water hydrogen-bond clusters. Regardless of the protonation state considered for PsbO, carboxylate pairs that can sample direct hydrogen bonding, or bridge via short hydrogen-bonded water chains, anchor to nearby basic or polar protein sidechains. As a result, carboxylic sidechains of the hypothesized antenna cluster are part of dynamic hydrogen bond networks that may rearrange rapidly when the protonation changes.     

Studies on the protonation constants and solvation of alpha-amino acids in dioxan-water mixtures

To gain more information about the effect of solvent on alpha-amino acids, the stoichiometric protonation constants of 10 alpha-amino acids (glycine, DL-alanine, DL-valine, L-leucine, L-isoleucine, DL-phenylalanine, L-serine, L-threonine, L-asparagine, and L-glutamine) in different dioxan-water mixtures have been determined potentiometrically using a combined pH electrode system calibrated in concentration units of hydrogen ion at 25 degrees C with an ionic strength of 0.10 M. For all amino acids studied, it was observed that the log K(1) values relating to the protonation equilibria of the anionic form are almost unaltered by the change in solvent composition. However, the log K(2) values corresponding to the formation equilibria of cationic form increase with the increase in dioxan content. The variation of these constants is discussed on the basis of specific solute-solvent interactions and structural changes of amino acids from water to dioxan-water media. The zwitterionic to neutral form ratio of these acids in dioxan-water mixtures is also discussed.     

Reprint of PSII Manganese Cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn₄CaO₅-cluster (Mn-cluster) in four discrete oxidation steps [S₁ − (S₄ / S₀)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Influence of membrane potentials upon reversible protonation of acidic residues from the OmpF eyelet

In this research we employed single-molecule electric recording techniques to investigate effects of the transmembrane and dipole potential on the reversible protonation of acidic residues from the constriction zone of the OmpF porin. Our results support the paradigm according to which the protonation state of aspartate 113 and glutamate 117 residues from the constriction region of OmpF is influenced by the electric potential profile, via an augmentation of the local concentration of protons near these residues mediated by increasing negative transmembrane potentials. We propose that at constant bulk pH, pK_a values for proton bindings at these residues increase as the applied transmembrane potential increases in its negative values. Our data demonstrate that the apparent pK_a for proton binding of the acidic aminoacids from the constriction region of OmpF is ionic strength-dependent, in the sense that a low ionic strength in the aqueous phase promotes the increase of the protonation reaction rate of such residues, at any given holding potential. Supplementary, we present evidence suggesting that lower values of the membrane dipole potential lead to an increase in the values of the ‘on’ rate of the eyelet acidic residues protonation, caused by an elevation of the local concentration of hydrogen ions. Altogether, these results come to support the paradigm according to which transmembrane and dipole potentials are critical parameters for the titration behavior of protein sites embedded lipid membranes.     

PSII manganese cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn₄CaO₅-cluster (Mn-cluster) in four discrete oxidation steps [S₁ − (S₄ / S₀)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Theoretical proton affinities of alpha1 adrenoceptor ligands

A systematic study has been performed of the proton affinity of a large family of agonists and antagonists of the alpha1-adrenoceptor at the B3LYP/6-31G* level of theory. After a conformational search, all the N atoms were considered as protonation sites and protonation energy values were determined. The inclusion of solvation by means of the Onsager model yielded stabilization in the proton affinity values obtained. In addition, a good correlation was found between the proton affinity values corresponding to the first protonation in gas phase of some of the compounds and their corresponding experimental affinity constants K(i) for the alpha1A adrenergic receptor.     

Proton transfer reaction and intermolecular interactions in associates of 2,5-dihydroxy-1,8-naphthyridine

Tautomerism in monomers/dimers and association of 2,5-dihydroxy-1,8-naphthyridine was studied at the DFT level recently recommended for studies of non-covalent interactions. Studied dimers are stabilized by double and triple hydrogen-bonding. In some associates the intermolecular proton transfer may take place. Transition state related to the double proton transfer reactions were calculated and discussed in terms of energetics, changes in atomic charges upon association, aromaticity (HOMA), properties of hydrogen bond critical point (QTAIM methodology) and geometry change during this reaction. It was found that double proton transfer is supported by third hydrogen bond or by weak secondary interaction. Some protons in transition states are shared between two basic atoms, while other are covalently bound only to one of them. The said process leads to replacement of secondary interactions of attractive character to repulsive and vice versa. Overall, results suggest that in subjected compound the triple hydrogen-bonded associate may be in equilibrium with double hydrogen-bonded dimer.     

Ionization of clusters. III. Evaluation of interaction parameters from binding data

Interactions between ionizable groups on the same molecule modulate the binding of protons to an extent where the binding constants may be shifted by orders of magnitude. The first two papers of this series discussed the family of carboxylic acids, pairwise isotropic interactions, and evaluation of single site binding data. This paper presents an extended group of hypothetical binding isotherms. Procedures are illustrated for deriving interaction parameters from binding data. The interaction parameters for about 25 representative compounds with two and three interacting ionizable groups are evaluated and tabulated.     

Effect of temperature and pressure on the protonation of glycine

Flow calorimetry has been used to study the interaction of glycine with protons in water at temperatures of 298.15, 323.15, and 348.15 K and pressures up to 12.50 MPa. By combining the measured heat for glycine solutions titrated with NaOH with the heat of ionization for water, the enthalpy of protonation of glycine is obtained. The reaction is exothermic at all temperatures and pressures studied. The effect of pressure on the enthalpy of reaction is very small. The experimental heat data are analyzed to yield equilibrium constant (K), enthalpy change (ΔH), and entropy change (ΔS) values for the protonation reaction as a function of temperature. These values are compared with those reported previously at 298.15 K. The ΔH and ΔS values increase (become more positive), whereas log K values decrease, as temperature increases. The trends for ΔH and ΔS with temperature are opposite to those reported previously for the protonation of several alkanolamines. However, log K values for proton interaction with both glycine and the alkanolamines decrease with increasing temperature. The effect of the nitrogen atom substituent on log K for protonation of glycine and alkanolamines is discussed in terms of changes in long-range and short-range solvent effects. These effects are used to explain the difference in ΔH and ΔS trends between glycine protonation and those found earlier for alkanolamine protonation.     

The ionization of clusters. II. Pairwise isotropic interactions and individual site binding data

Evaluation of the parameters describing the binding of protons to clusters of interacting sites requires some reasonable assumptions and procedures because it is impossible to observe an unperturbed site in its interacting environment. When the unperturbed sites are not identical, individual site binding data allow for the evaluation of the differences (or ratios) between the unperturbed (or intrinsic) binding constants but not their actual values (or the interaction energies). In this paper we extend our previous treatment of the ionization of clusters in order to generalize pairwise isotropic interactions and take into account the present availability of individual site binding data.     

Energetics of ribonuclease A catalysis. 3. Temperature dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

Studies of the temperature dependence of the steady-state kinetics of the ribonuclease A catalyzed hydrolysis of cytidine cyclic 2',3'-phosphate at pH 5.0 are reported. Contributions to the temperature dependence of the apparent Michaelis-Menten parameters from temperature-sensitive protonic equilibria (primarily the coupled protonation/deprotonation of the active-site histidine residues) were included in our analysis. The data were interpreted by employing a transition-state approach. By comparing the temperature dependence of the rate constant for the nonenzymatic hydrolysis of the substrate with the temperature dependence of the enzyme-catalyzed reaction, we obtained values for the enthalpy change, entropy change, and heat capacity change for the interaction of the reaction transition state with the enzyme. These thermodynamic quantities were then interpreted by comparison with corresponding values for the binding of cytidine 2'- and 3'-phosphate to the enzyme. A model is presented for the enzyme-transition-state interaction involving the favorable transfer of a proton from the transition state to a histidine residue at the active site and the formation of hydrogen bonds and van der Waals contacts between the pyrimidine ring of the transition state and the enzyme's binding pocket. These elementary interactions are consistent with the determined values of the enthalpy change and entropy change, as well as earlier reported ionic strength and solvent isotope dependence studies. The Gibbs energy contributions from these elementary interactions have also been estimated, giving a sum approximately equal to the experimentally determined value for the stabilization energy of the enzyme-transition-state complex. The model thus provides an explanation for the magnitude of the approximately 10(10)-fold rate enhancement achieved by this enzyme.     

15N- and 1H-NMR investigations of the active-site amino acids in semisynthetic RNase S' and RNase A

Extensive 15N-NMR investigations of active-site amino acids were made possible by the solid-phase synthesis of the N-terminal pentadecapeptide of RNase A with selectively 15N-enriched amino acids. On complexation with S-protein a fully active RNase S' complex was obtained. The 15N resonances of the side chains of lysine-7 (N epsilon), glutamine-11 (N gamma), and histidine-12 (N pi, tau) were studied in the free synthetic peptide, in the RNase S' complex and in the nucleotide complexes RNase S' with 2'CMP, 3'CMP, and 5'AMP. The analysis of the 15N-1H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide-protein interaction, the side chains of Lys-7 and Gln-11 do not contribute to this interaction. In the nucleotide complexes the resonances of His-12 and Gln-11 are shifted downfield. In the 2'CMP complex a doublet for the N tau signal of His-12 indicates a stable H bond between this nitrogen and the phosphate group of nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln-11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys-7 signal is line broadening due to reduced proton exchange. For comparison with the 15N-NMR titration curves of His-12 in RNase S' the 1H-NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the 15N and the 1H titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active-site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the 15N resonances, show a small inflection in the pH range 5.8-6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active-site histidines by a partial H-bond formation between the imidazole groups.