Protonation State Research Articles

Phosphoester bond hydrolysis by a discrete zirconium-oxo cluster: mechanistic insights into the central role of the binuclear ZrIV-ZrIV active site.

Effective degradation of non-natural phosphate triesters (PTs) widely used in pesticides and warfare agents is of paramount relevance for human and environmental safety, particularly under acidic conditions where they are highly stable. Here, we present a detailed reactivity and mechanistic study pioneering discrete {Zr6O8} clusters, which are commonly employed as building blocks for Zr-MOFs and as non-classical soluble coordination compounds for the degradation of PTs using the pesticide ethyl paraoxon as a model. Combined computational studies, mechanistic experiments, and EXAFS analysis show that the reactivity of these clusters arises from their ZrIV-ZrIV bimetallic sites, which hydrolyze ethyl paraoxon under acidic conditions through an intramolecular pathway. Remarkably, the energetics of the reaction is dependent on the protonation state of the active sites, and a weakly acidic medium favors the reaction. Moreover, catalyst stability allowed for its recovery and reuse. Such a mechanism is in close analogy to enzymatic reactions and different from that previously reported for Zr-MOFs. These findings outline the potential of MIV-MIV active sites for PT degradation under challenging aqueous acidic conditions and contribute to the development of bioinspired catalysts and materials.

Measuring local pH at interfaces from molecular tumbling: A concept for designing EPR-active pH-sensitive labels and probes.

Molecular probes and indicators are broadly employed for pH measurements in bulk media and at interfaces. The underlying physical principle of pH measurements of most of these probes is based on a change in the electronic structure that, for example, results in a shift of the emission peak of the fluorescence probes, changes in NMR chemical shifts due to the affected electronic shielding, or magnetic parameters of pH-sensitive nitroxides as measured by EPR. Here we explore another concept for measuring local protonation state of molecular tags based on changes in rotational dynamics of electron spin-bearing moieties that are readily detected by conventional continuous wave X-band EPR. Such changes are especially pronounced at biological interfaces, such as lipid bilayer membranes, due to the probe interactions with adjacent charges and polarizable dipoles. The concept was demonstrated by synthesizing a series of pH-sensitive nitroxides and spin-labelled phospholipids. EPR spectra of these newly synthesized nitroxides exhibit relatively small - about 0.5 G - changes in isotropic nitrogen hyperfine coupling constant upon reversible protonation. However, spin-labelled phospholipids incorporated into lipid bilayers demonstrated almost 6-fold change in rotational correlation time upon protonation, readily allowing for pKa determination from large changes in EPR spectra. The demonstrated concept of EPR-based pH measurements leads to a broader range of potential nitroxide structures that can serve as molecular pH sensors at the desired pH range and, thus, facilitates further development of spin-labelling EPR methods to study electrostatic phenomena at chemical and biological interfaces.

Exploiting the full potential of cryo-EM maps

The Coulomb potential maps generated by electron microscopy (EM) experiments contain not only information about the position but also about the charge state of the atom. This feature of EM maps allows the identification of specific ions and the protonation state of amino acid side chains in the sample. Here, we summarize qualitative observations of charges in EM maps, discuss the difficulties in interpreting the charge in Coulomb potential maps with respect to distinguishing it from radiation damage, and outline considerations to implement the correct charge in fitting algorithms.

New model for aspartic acid species in aqueous calcium carbonate growth environments: challenges and perspectives.

The lack of experimental data on the dynamics of aspartic acid species in water for its range of protonation states and the details of their atomic-level interaction with aqueous calcium carbonate species is a driver for accurate force field development. A classical model that is consistent with the few pieces of experimental data available and with first principles calculations has been developed. The complex dynamics of the aspartate anions relevant to biomineralization and calcium carbonate crystal growth has been explored in water, providing a quantitative description of solvation structure and free energies, including conformational free energy profiles and pairing free energies. The model has been used to probe the structure and dynamics of aqueous calcium aspartate homo- and hetero-chiral clusters, confirming their unlikelihood due to weak and water-mediated interactions. This supports the hypothesis that the formation of such clusters, observed while growing vaterite in the presence of acidic chiral amino acids, is favoured by the presence of the crystal surface.

A new biphenol-dipicolylamine based ligand and its dinuclear Zn2+ complex as fluorescent sensors for ibuprofen and ketoprofen in aqueous solution.

In this work, the study of the new ligand 3,3'-bis[N,N-bis(pyridine-2-ylmethyl)aminomethyl]-2,2'-dihydroxybiphenyl (L) is reported, where a central 2,2'-biphenol (BPH) fluorophore was functionalized at 3,3'-positions with two dipicolylamine (DPA) side arms as receptor units. Following the synthesis and full chemical-physical characterization, the acid-base and Zn2+-coordination abilities of L were investigated through a combination of potentiometric, UV-Vis, fluorescence, NMR, XRD and DFT measurements. The optical properties of the ligand turned out to be strongly dependent on the pH, being straightforwardly associated with the protonation state of the BPH moiety, whereas its peculiar design allowed to form stable mono and dinuclear Zn2+ complexes. In the latter species, the presence of two Zn2+ ions coordinatively unsaturated and placed at close distance to each other, prompted us to test their usefulness as metallo-receptors for two environmental pollutants of great relevance, ibuprofen and ketoprofen. Potentiometric and fluorescence investigations evidenced that these important non-steroidal anti-inflammatory drugs (NSAIDs) are effectively coordinated by the metallo-receptors and, of relevance, both the stability and the fluorescence properties of the resulting ternary adducts are markedly affected by the different chemical architectures of the two substrates. This study aims at highlighting the promising perspectives arising from the use of polyamino phenolic ligands as chemosensors for H+/Zn2+ and other additional anionic targets in their metal-complexed forms.

An albumin unfolding and refolding cycle induced by a time-controlled pH jump.

Given the intimate connection between the structure and function of biological macromolecules, the ability to temporally control their unfolding-refolding process enables temporal regulation over specific functionalities, potentially applicable in innovative domains, including the construction of protein-based actuators or programmable catalysis and drug release in complex biotechnological processes. We show here how a temporally controlled protein unfolding-refolding cycle can be coupled in time with programmed pH sequences achieved through the spontaneous decomposition of an activated carboxylic acid. Specifically, we illustrate this process at the molecular level using albumin, the most prevalent protein found in plasma, for which a temporary shift from native to unfolded forms is promoted using nitroacetic acid, able to undergo base-catalysed decarboxylation when solubilized in water solution. As detected by small angle X-ray scattering and intrinsic tryptophan fluorescence, starting from the protein in its native form, the acid addition triggers unfolding to a partially denatured state and a subsequent time-tunable pH rise with gradual refolding that recapitulates the intermediate steps detected at the same pH values by static acidification, fitting within a framework of full reversibility of the structural changes as a function of the protein protonation state. At the end of the pH jump, the native structure is fully recovered, making this method a chemical tool to achieve a complete protein conformational sequence programmed in the timeframe of minutes without further intervention.

Electronic state modulation of Ag30 nanoclusters within a ring-shaped polyoxometalate.

Atomically precise Ag nanoclusters display distinctive properties that are dictated by their structures and electronic states. However, manipulating the electronic states of Ag nanoclusters is challenging owing to their inherent instability and susceptibility to undesired structural changes, decomposition, and aggregation. Recently, we reported the synthesis of a body-centered cubic {Ag30}22+ nanocluster encapsulated within a ring-shaped polyoxometalate (POM) [P8W48O184]40- by reacting 16 Ag+-containing [P8W48O184]40- with Ag+ using N,N-dimethylformamide (DMF) as a mild reducing agent. This led to a redox-induced structural transformation into a face-centered cubic {Ag30}16+ nanocluster. In this study, we demonstrated the modulation of the electronic states of Ag30 nanoclusters within the ring-shaped POM through two different approaches. A face-centered cubic {Ag30}18+ nanocluster, featuring distinct oxidation states compared to previously reported {Ag30}22+ and {Ag30}16+ nanoclusters, was synthesized using tetra-n-butylammonium borohydride, a stronger reducing agent than DMF, in the reaction of 16 Ag+-containing [P8W48O184]40- and Ag+. Additionally, by leveraging the acid-base properties of POMs, we demonstrated the reversible, stepwise modulation of the charge distribution in the Ag30 nanocluster through controlling protonation states of the ring-shaped POM ligand. These results highlight the potential of engineering POM-stabilized Ag nanoclusters with diverse structures and electronic states, thereby facilitating the exploration of novel properties and applications utilizing the unique characteristics of the POM ligands.

Structural descriptors and information extraction from X-ray emission spectra: aqueous sulfuric acid.

Machine learning can reveal new insights into X-ray spectroscopy of liquids when the local atomistic environment is presented to the model in a suitable way. Many unique structural descriptor families have been developed for this purpose. We benchmark the performance of six different descriptor families using a computational data set of 24 200 sulfur Kβ X-ray emission spectra of aqueous sulfuric acid simulated at six different concentrations. We train a feed-forward neural network to predict the spectra from the corresponding descriptor vectors and find that the local many-body tensor representation, smooth overlap of atomic positions and atom-centered symmetry functions excel in this comparison. We found a similar hierarchy when applying the emulator-based component analysis to identify and separate the spectrally relevant structural characteristics from the irrelevant ones. In this case, the spectra were dominantly dependent on the concentration of the system, whereas adding the second most significant degree of freedom in the decomposition allowed for distinction of the protonation state of the acid molecule.

Experimental and theoretical studies of pH-responsive iridium(III) complexes of azole and N-heterocyclic carbene ligands.

A series of nine luminescent iridium(III) complexes with pH-responsive imidazole and benzimidazole ligands have been prepared and characterized. The first series of complexes were of the form [Ir(ppy)2(N^N)]+ or [Ir(ppy)2(C^N)]+ (where ppy is 2-phenylpyridine and N^N is 2-(2-pyridyl)imidazole or 2-(2-pyridyl)benzimidazole and C^N represents a pyridyl-triazolylidene-based N-heterocyclic carbene ligand). For these complexes, the benzimidazole group was either unsubstituted or substituted with electron-withdrawing (Cl) or electron-donating (Me) groups. The second series of complexes were of the form [Ir(phbim)2(N^N)]+ or [Ir(phbim)2(C^N)]+ (where phbim is 2-phenylbenzimidazole and N^N is either 2,2'-bipyridine or 1,10-phenanthroline and C^N is either a pyridyl-imidazolylidene or pyridyl-triazolylidene N-heterocyclic carbene ligand). UV-visible and photoluminescence pH titration studies showed that changing the protonation state of these complexes results in significant changes in the photoluminescence emission properties. The pKa values of prepared complexes were estimated from the spectroscopic pH titration data and these values show that the nature of the pH-sensitive ligands (either main or ancillary ligands) resulted in a significant capacity to modulate the pKa values for these compounds with values ranging from 5.19-11.22. Theoretical investigations into the nature of the electronic transitions for the different protonation states of compounds were performed and the results were consistent with the experimental results.

Tuning Potential Functions to Host-Guest Binding Data.

Software to more rapidly and accurately predict protein-ligand binding affinities is of high interest for early-stage drug discovery, and physics-based methods are among the most widely used technologies for this purpose. The accuracy of these methods depends critically on the accuracy of the potential functions that they use. Potential functions are typically trained against a combination of quantum chemical and experimental data. However, although binding affinities are among the most important quantities to predict, experimental binding affinities have not to date been integrated into the experimental data set used to train potential functions. In recent years, the use of host-guest complexes as simple and tractable models of binding thermodynamics has gained popularity due to their small size and simplicity, relative to protein-ligand systems. Host-guest complexes can also avoid ambiguities that arise in protein-ligand systems such as uncertain protonation states. Thus, experimental host-guest binding data are an appealing additional data type to integrate into the experimental data set used to optimize potential functions. Here, we report the extension of the Open Force Field Evaluator framework to enable the systematic calculation of host-guest binding free energies and their gradients with respect to force field parameters, coupled with the curation of 126 host-guest complexes with available experimental binding free energies. As an initial application of this novel infrastructure, we optimized generalized Born (GB) cavity radii for the OBC2 GB implicit solvent model against experimental data for 36 host-guest systems. This refitting led to a dramatic improvement in accuracy for both the training set and a separate test set with 90 additional host-guest systems. The optimized radii also showed encouraging transferability from host-guest systems to 59 protein-ligand systems. However, the new radii are significantly smaller than the baseline radii and lead to excessively favorable hydration free energies (HFEs). Thus, users of the OBC2 GB model currently may choose between GB cavity radii that yield more accurate binding affinities and GB cavity radii that yield more accurate HFEs. We suspect that achieving good accuracy on both will require more far-reaching adjustments to the GB model. We note that binding free-energy calculations using the OBC2 model in OpenMM gain about a 10× speedup relative to corresponding explicit solvent calculations, suggesting a future role for implicit solvent absolute binding free-energy (ABFE) calculations in virtual compound screening. This study proves the principle of using host-guest systems to train potential functions that are transferrable to protein-ligand systems and provides an infrastructure that enables a range of applications.

Determination of the relative sensitivity of polyaniline inks and films to deprotonation by ammonia using UV–Vis spectroscopy

The deprotonation behavior of conducting polymers such as polyaniline (PANI) is an important functional parameter in the design of novel printed electronic devices, particularly for sensors and energy storage. PANI can be fabricated into films from precursor liquid-processable ‘ink’ formulations. The effective determination and comparison of the deprotonation behavior of such inks is critical to understanding their subsequent functional performance. While protonation can be measured by titration with protonating and deprotonating agents using UV–vis spectroscopy, comparative analysis has proven challenging due to complexities in the measurement of polymer concentration and consequent lack of comparability. This work introduced a coherent approach to establishing the arbitrary concentration of PANI ink formulations. Dodecylbenzene sulfonic acid (DBSA)-micellar doped PANI nanoparticles suspended in sodium dodecyl sulfate were prepared using various monomer, oxidant, and dopant ratios, titrated with ammonia and analyzed using UV–vis spectroscopy. Isosbestic points unaffected by doping or protonation state were used to establish arbitrary polymer concentration. Similar titrations on films prepared from these formulations were performed using ammonia in the gas phase and were based on calculations of deposited mass. Parameters such as a ‘doping capacity’, and their relative sensitivity to ammonia could be determined, which could in turn be related to the physical and chemical compositions of the resulting inks and films. The developed methodology has the potential to be a simple and convenient means with which to compare and optimize the functional deprotonation behavior of conducting polymer inks and films for device development and production optimization.

Probing the role of the protonation state of a minor groove-linker histidine in Exd-Hox–DNA binding

DNA recognition and targeting by transcription factors (TFs) through specific binding are fundamental in biological processes. Furthermore, the histidine protonation state at the TF-DNA binding interface can significantly influence the binding mechanism of TF-DNA complexes. Nevertheless, the role of histidine in TF-DNA complexes remains underexplored. Here, we employed all-atom molecular dynamics simulations using AlphaFold2-modeled complexes based on previously solved co-crystal structures to probe the role of the His-12 residue in the Extradenticle (Exd)-Sex combs reduced (Scr)-DNA complex when binding to Scr and Ultrabithorax (Ubx) target sites. Our results demonstrate that the protonation state of histidine notably affected the DNA minor-groove width profile and binding free energy. Examining flanking sequences of various binding affinities derived from SELEX-seq experiments, we analyzed the relationship between binding affinity and specificity. We uncovered how histidine protonation leads to increased binding affinity but can lower specificity. Our findings provide new mechanistic insights into the role of histidine in modulating TF-DNA binding.

Protomer of Imipramine Captured in Cucurbit[7]uril.

Small molecules possessing multiple proton-accessible sites are important not only to many biological systems but also to host-guest chemistry; their protonation states are causal to boosting or hindering specific host-guest interactions. However, determining the protonation site is not trivial. Herein, we conduct electrospray ionization ion mobility spectrometry-mass spectrometry to imipramine, a known molecule with two protonation sites, based on the introduction of cucurbit[7]uril as a host molecule. For protonated imipramine, the proposed strategy allows clear distinction of the two protomers as host-guest complex ions and can be leveraged to capture the energetically less preferable protomer of the protonated imipramine.

Inhibition mechanism understanding from molecular dynamics simulation of the interactions between several flavonoids and proton-dependent glucose transporter

Proton-dependent glucose transporters as important drug targets can have different protonation states and adjust their conformational state under different pHs. So based on this character, research on its inhibition mechanism is a significant work. In this article, to study its inhibitory mechanism, we performed the molecular dynamics of several classical flavonoid molecules (Three inhibitors Phloretin, Naringenin, Resveratrol. Two non-inhibitors Isoliquiritigenin, Butein) with glucose transporters under two distinct environmental pHs. The results show inhibitors occupy glucose binding sites (GLN137, ILE255, ASN256) and have strong hydrophobic interactions with proteins through core moiety (C6-Cn-C6). In addition, inhibitors had better inhibitory effects in protonation state. In contrast, non-inhibitors can not occupy glucose binding sites (GLN137, ILE255, ASN256), thus they do not have intense interactions with the protein. It is suggested that favorable inhibitors should effectively take up the glucose-binding site (GLN137, ILE255, ASN256) and limit the protein conformational changes.Communicated by Ramaswamy H. Sarma

Development of Mefloquine-Based Bifunctional Secondary Amine Organocatalysts for Enantioselective Michael and Friedel-Crafts Reactions.

The chiral framework based on 11-aminomefloquine has been utilized for the first time to construct bifunctional organocatalysts. These catalysts demonstrate high enantioselectivity in both Michael additions and Friedel-Crafts reactions across a variety of substrates, achieving up to >99% ee. The distinctive feature is the incorporation of a secondary amine group, offering unique tight hydrogen-bonding capabilities in the protonated state, as supported by DFT computation. The diversity of these organocatalysts suggests their broad applicability across multiple reaction classes.

The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces.

The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.

Solvent Effects and pH Dependence of the X-ray Absorption Spectra of Proline from Electrostatic Embedding Quantum Mechanics/Molecular Mechanics and Mixed-Reference Spin-Flip Time-dependent Density-Functional Theory.

The accurate description of solvent effects on X-ray absorption spectra (XAS) is fundamental for comparing the simulated spectra with experiments in solution. Currently, few protocols exist that can efficiently reproduce the effects of the solute/solvent interactions on XAS. Here, we develop an efficient and accurate theoretical protocol for simulating the solvent effects on XAS. The protocol combines electrostatic embedding QM/MM based on electrostatic potential fitted operators for describing the solute/solvent interactions and mixed-reference spin-flip time-dependent density functional theory (MRSF-TDDFT) for simulating accurate XAS spectra. To demonstrate the capabilities of our protocol, we compute the X-ray absorption of neutral proline in the gas phase and ionic proline in water in all relevant K-edges, showing excellent agreement with experiments. We show that states represented by core to π* transitions are almost unaffected by the interaction with water, whereas the core to σ* transitions are more impacted by the fluctuation of proline structure and the electrostatic interaction with the solvent. Finally, we reconstruct the pH-dependent XAS of proline in solution, determining that the N K-edge can be used to distinguish its three protonation states.

Chemical complexity challenge: Is multi‐instance machine learning a solution?

AbstractMolecules are complex dynamic objects that can exist in different molecular forms (conformations, tautomers, stereoisomers, protonation states, etc.) and often it is not known which molecular form is responsible for observed physicochemical and biological properties of a given molecule. This raises the problem of the selection of the correct molecular form for machine learning modeling of target properties. The same problem is common to biological molecules (RNA, DNA, proteins)—long sequences where only key segments, which often cannot be located precisely, are involved in biological functions. Multi‐instance machine learning (MIL) is an efficient approach for solving problems where objects under study cannot be uniquely represented by a single instance, but rather by a set of multiple alternative instances. Multi‐instance learning was formalized in 1997 and motivated by the problem of conformation selection in drug activity prediction tasks. Since then MIL has found a lot of applications in various domains, such as information retrieval, computer vision, signal processing, bankruptcy prediction, and so on. In the given review we describe the MIL framework and its applications to the tasks associated with ambiguity in the representation of small and biological molecules in chemoinformatics and bioinformatics. We have collected examples that demonstrate the advantages of MIL over the traditional single‐instance learning (SIL) approach. Special attention was paid to the ability of MIL models to identify key instances responsible for a modeling property.This article is categorized under: Data Science > Chemoinformatics Data Science > Artificial Intelligence/Machine Learning

PH-Dependent Structure and Dynamics of the Catalytic Domains of Human Carbonic Anhydrase II and IX.

Extensive computer simulation studies have been carried out to probe the pH-dependent structure and dynamics of the two most efficient isoenzymes II and IX of human carbonic anhydrase (HCA) that control the pH in the human body. The equilibrium structure and hydration of their catalytic domains are found to be largely unaffected by the variation of pH in the range studied, in close agreement with the known experimental results. In contrast, a significant effect of the change in pH is observed for the first time on the local electrostatic potential of the active site walls and the dynamics of active site water molecules. We also report for the first time the free energy and kinetics of coupled fluctuations of orientation and protonation states of the well-known His-mediated proton shuttle (His-64) in both isozymes at pH 7 and 8. The transitions between different tautomers of in or out conformations of His-64 side chain range between 109 and 106 s-1 depending on pH. Possible implications of these results on conformation-dependent pKa of His-64 side chain and its role in driving the catalysis toward hydration of CO2 or dehydration of HCO3- with varying pH are discussed.

Phage display assisted discovery of a pH-dependent anti-α-cobratoxin antibody from a natural variable domain library.

RecyclingIgG antibodies bind to their target antigen at physiological pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the IgG antibodies to be recycled into circulation via FcRn-mediated cellular pathways, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non-recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. Both are strategies that may introduce sequence liabilities. Here, we present a methodology that employs a naïve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind α-cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. As a result, an antibody was discovered that exhibits a 7-fold higher off-rate at pH 5.5 than pH 7.4 in bio-layer interferometry experiments. Interestingly, no histidine residues were found in its variable domains, and in addition, the antibody showed pH-dependent binding to a histidine-devoid antigen mutant. As such, the results demonstrate that pH-dependent antigen-antibody binding may not always be driven by histidine residues. By employing molecular dynamics simulations, different protonation states of titratable residues were found, which potentially could be responsible for the observed pH-dependent antigen binding properties of the antibody. Finally, given the typically high diversity of naïve antibody libraries, the methodology presented here can likely be applied to discover recycling antibodies against different targets ab initio without the need for histidine doping.