Protonation State Research Articles

Revisiting the influence of pH on 1JCαH and chemical shifts of glycine and alanine short oligopeptides.

The pH dependence of several NMR parameters of glycine and alanine short oligopeptides has been reported previously in different studies. Here we have thoroughly examined, summarized and demonstrated the dependences of 1H, 13C and 15N chemical shifts and protonation states of amino acids using two-dimensional NMR experiments. Nevertheless, 1JCαH one bond spin-spin coupling constants are more informative and convenient for determination of the position and protonation state of glycine and alanine residue in the oligopeptide chain. In particular, for various oligopeptides (up to six residues), it was shown that the pH dependence of 1JCαH of N-terminal glycine and alanine residues is larger than that of C-terminal groups, and in backbone residues, it is not influenced by pH and only slightly depends on the position of the amino acid residue in the chain.

Enhanced Uptake of Anticancer C6-Pep Dimer in a Model Membrane Caused by Differential pKa in Acidic Microenvironment.

Acidic tumor microenvironment (TME) presents a challenge for the action of antitumor drugs by acting as an additional barrier for the passive crossing of the cell membrane by chemotherapic agents playing a critical role in the proliferation of tumor cells. Anticancer lipopeptide C6-Pep dimer containing the leucine zipper motif shows an increased uptake into the model tumor membrane in TME, and application of external heat might lead to the uncoiling of the zipper, which could result in cell lysis. This work investigated the cause of this increased uptake of C6-Pep dimer into the bilayer model in TME. Accurate protonation states of all the titratable residues of the C6-Pep dimer in TME were determined using constant pH molecular dynamics. In TME, except for two terminal Glu5 residues, all other Glu residues in the C6-Pep dimer were permanently protonated. The remaining Glu5 residues had differential pKa values, leading to the construction of four possible dimers with different fixed protonation states, and molecular dynamics was used to study their interaction with the anionic bilayer. Except for the dimer at a physiological pH, the other dimers were positively charged and could readily adsorb on the membrane surface. The free energy of insertion of these dimers in the bilayer was lower for single and double protonated Glu5-containing dimers than for the others. After the insertion of the lipopeptides into the membrane, thinning of the bilayer in the vicinity of dimers and an increase in area per lipid of the bilayer were observed for all systems, indicating destabilization of the bilayer due to this intercalation. This study shows that the anticancer lipopeptide C6-Pep utilizes the TME around a tumor cell for insertion into the membrane.

Dynamic Protonation States Underlie Carbene Formation in ThDP-Dependent Enzymes: A Theoretical Study.

The activation mechanism of thiamine diphosphate (ThDP) in enzymes has long been the subject of intense research and controversial discussion. Particularly contentious is the formation of a carbene intermediate, the first one observed in an enzyme. For the formation of the carbene to take place, both intramolecular and intermolecular proton transfer pathways have been proposed. However, the physiologically relevant pH of ThDP-dependent enzymes around neutrality does not seem to be suitable for the formation of such reactive chemical species. Herein, we investigate the general mechanism of activation of the ThDP cofactor in human transketolase (TKT), by means of electronic structure methods. We show that in the case of the human TKT, the carbene species is accessible through a pKa shift induced by the electrostatics of a neighboring histidine residue (H110), whose protonation state change modulates the pKa of ThDP and suppresses the latter by more than 6 pH units. Our findings highlight that ThDP enzymes activate the cofactor beyond simple geometric constraints and the canonical glutamate. Such observations in nature can pave the way for the design of biomimetic carbene catalysts and the engineering of tailored enzymatic carbenes.

Examination of ‘high-energy’ metastable state of the oxidized (OH) bovine cytochrome c oxidase: Proton uptake and reaction with H2O2

Reoxidized cytochrome c oxidase appears to be in a ‘high-energy’ metastable state (OH) in which part of the energy released in the redox reactions is stored. The OH is supposed to relax to the resting ‘as purified’ oxidized state (O) in a time exceeding 200 ms. The catalytic heme a3-CuB center of these two forms should differ in a protonation and ligation state and the transition of OH-to-O is suggested to be associated with a proton transfer into this center. Employing a stopped-flow and UV–Vis absorption spectroscopy we investigated a proton uptake during the predicted relaxation of OH. It is shown, using a pH indicator phenol red, that from the time when the oxidation of the fully reduced CcO is completed (∼25 ms) up to ∼10 min, there is no uptake of a proton from the external medium (pH 7.8). Moreover, interactions of the assumed OH, generated 100 ms after oxidation of the fully reduced CcO, and the O with H2O2 (1 mM), result in the formation of two ferryl intermediates of the catalytic center, P and F, with very similar kinetics and the amounts of the formed ferryl states in both cases. These results implicate that the relaxation time of the catalytic center during the OH-to-O transition is either shorter than 100 ms or there is no difference in the structure of heme a3-CuB center of these two forms.

Model-Assisted Interpretation of 4,4'-Methylene Dianiline Adsorption on Soils at Micromolar Concentrations.

Adsorption kinetics and isotherms were determined for 4,4'-methylene dianiline (MDA) on five diverse soils at nominal concentrations of 0.01-1.0 mg L-1 (nominal soil loading 0.1-40 μg gs -1 ). The data were used to model the adsorption process based on the two-step mechanism that is characteristic of the adsorption of aromatic amines, consisting of a physical equilibrium between the aqueous phase and the soil organic matter and a chemical reaction between the adsorbed MDA and reactive sites in the soil organic matter. Generic parameters were determined that enabled application of the model to other soils, which was checked against previously published data for MDA adsorption. At the low concentrations evaluated, the adsorption process took place almost exclusively in the organic matter without the need to account for a separate ion exchange process with the soil mineral fraction. Physical adsorption was found to be mainly dependent on the protonation state of MDA and increased with decreasing pH of the soils. Because of the chemical reaction taking place, adsorption equilibrium constants (organic-carbon partition coefficient [KOC ]) normalized to the organic carbon content in the soil gradually increased with time; and it was demonstrated that, at steady-state conditions, values of log KOC > 3.5 can be expected for most any soil at conservatively estimated potential environmental MDA concentrations. Environ Toxicol Chem 2023;42:2580-2588. © 2023 SETAC.

Chitosan Polysaccharides from a Polarizable Multiscale Approach.

We report simulations of chitosan polysaccharides in the aqueous phase, at infinite dilute conditions and zero ionic strength. Those simulations are performed by means of a polarizable multiscale modeling scheme that relies on a polarizable all atom force field to model solutes and on a polarizable solvent coarse grained approach. Force field parameters are assigned only from quantum chemistry ab initio data. We simulate chitosan monomer units, dimers and 50-long chains. Regarding the 50-long chains we simulate three sets of ten randomly built chain replica at three different pH conditions (corresponding to different chain protonation states, the chain degree of deacetylation is 85%). Our simulations show the persistence length of 50-long chitosan chains at strong acidic conditions (pH <5) to be 24 ± 2 nm (at weak/negligible ionic strength conditions), and to be 1 order of magnitude shorter at usual pH conditions. Our simulation data support the most recent simulation and experimental studies devoted to chitosan polysaccharides in the aqueous phase.

Cover Feature: Monitoring Conformation and Protonation States of Glutathione by Raman Optical Activity and Molecular Dynamics (ChemPlusChem 11/2023)

Cover Feature: Monitoring Conformation and Protonation States of Glutathione by Raman Optical Activity and Molecular Dynamics (ChemPlusChem 11/2023)

Aggregation-Induced Asymmetric Charge States of Amino Acids in Supramolecular Nanofibers.

Electrostatic interactions contribute critically to the kinetic pathways and thermodynamic outcomes of peptide self-assembly involving one or more than one charged amino acids. While it is well understood in protein folding that those amino acids with acidic/basic side chains could shift their pKas when placed in a hydrophobic microenvironment, to what extent aggregation of monomeric peptide units from the bulk solution could alter their charged status and how this change in pKa values would reciprocally impact their assembly outcomes. Here, we design and analyze two solution systems containing peptide amphiphiles with hydrocarbon chains of different lengths to determine the factor of deprotonation on assembly. Our results suggest that models of supramolecular nanofibers with uniformly distributed, fully charged amino acids are oversimplified. We demonstrate, with molecular dynamics simulations, and validate with experimental results that asymmetric, different protonation states of the peptides lead to distinct nanostructures after self-assembly. The results give estimates on the electrostatic interactions in peptide amphiphiles required for their self-assembly and shed light on modeling molecular assembly systems containing charged amino acids.

Diverse Coordination Geometries Derived from Trisaminocyclohexane Ligands with Appended Outer‐Sphere Hydrogen Bond Donors

AbstractWith the aim of constructing hydrogen‐bonding networks in synthetic complexes, two new ligands derived from cis,cis‐1,3,5‐triaminocyclohexane (TACH) have been prepared that feature pendant pyrrole or indole rings as outer‐sphere H‐bond donors. The TACH framework offers a facial arrangement of three N‐donors, thereby mimicking common coordination motifs in the active sites of nonheme Fe and Cu enzymes. X‐ray structural characterization of a series of CuI‐X complexes (X=F, Cl, Br, NCS) revealed that these neutral ligands (H3LR, R=pyrrole or indole) coordinate in the intended facial N3 manner, yielding four‐coordinate complexes with idealized C3 symmetry. The N−H units of the outer‐sphere heterocycles form a hydrogen‐bonding cavity around the axial (pseudo)halide ligand, as verified by crystallographic, spectroscopic, and computational analyses. Treatment of H3Lpyrrole and H3Lindole with divalent transition metal chlorides (MIICl2, M=Fe, Cu, Zn) causes one heterocycle to deprotonate and coordinate to the M(II) center, giving rise to tetradentate ligands with two remaining outer‐sphere H‐bond donors. Further ligand deprotonation is observed upon reaction with Ni(II) and Cu(II) salts with weakly coordinating counteranions. The reported complexes highlight the versatility of TACH‐based ligands with pendant H‐bond donors, as the resulting scaffolds can support multiple protonation states, coordination geometries, and H‐bonding interactions.

PLP-Dependent Enzyme Methionine γ-Lyase: Insights into the Michaelis Complex from Molecular Dynamics and Free Energy Simulations.

Methionine γ-lyase (MGL) breaks down methionine, with the help of its cofactor pyridoxal-5'-phosphate (PLP), or vitamin B6. Methionine depletion is damaging for cancer cells but not normal cells, so MGL is of interest as a therapeutic protein. To increase our understanding and help engineer improved activity, we focused on the reactive, Michaelis complex between MGL, covalently bound PLP, and substrate Met. is not amenable to crystallography, as it proceeds to products. Experimental activity measurements helped exclude a mechanism that would bypass . We then used molecular dynamics and alchemical free energy simulations to elucidate its structure and dynamics. We showed that the PLP phosphate has a pKa strongly downshifted by the protein, whether Met is present or not. Met binding affects the structure surrounding the reactive atoms. With Met, the Schiff base linkage between PLP and a nearby lysine shifts from a zwitterionic, keto form to a neutral, enol form that makes it easier for Met to approach its labile, target atom. The Met ligand also stabilizes the correct orientation of the Schiff base, more strongly than in simulations without Met, and in agreement with structures in the Protein Data Bank, where the Schiff base orientation correlates with the presence or absence of a co-bound anion or substrate analogue in the active site. Overall, the Met ligand helps organize the active site for the enzyme reaction by reducing fluctuations and shifting protonation states and conformational populations.

Charge transport in protic ionic liquids: Effect of protonation state in 1-methylimidazolium – acetate/trifluoroacetate mixtures

Despite the potential to overcome slow charge transport in ionic liquids by decoupling charge transport from mass transport in protic ionic liquids (PILs), the average proton distribution and the contribution of proton conduction to the overall charge transport in PILs has not been fully established. To elucidate the predominant underlying molecular species in the PILs and the molecular dynamics of the PILs, we study the dipolar response and conductivity using dielectric relaxation (DR) spectroscopy and the nucleis chemical environment and molecular mobility using nuclear magnetic resonance (NMR) spectroscopy of prototypical PILs based on 1–methylimidazolium as Brønsted base. To elucidate the effect of the acidity of the Brønsted acid we gradually vary the composition of the PILs from 1–methylimidazoliumacetate [MimH][AcO] to 1–methylimidazoliumtrifluoroacetate [MimH][TFA]. Both, 15N-NMR chemical shifts and the sample conductivities suggest that in neat [MimH][AcO] electro-neutral molecular species dominate, consistent with earlier findings. The dipolar response of these molecular species as detected via the DR spectra is stronger than what one would expect based on the molecular dipole moments estimated from density functional theory calculations, which points to the formation of an extended hydrogen-bonded network. Upon addition of [MimH][TFA], this dipolar response is reduced as the degree of protonation of 1–methylimidazolium gradually increases, which is apparent from the conductance and 15N chemical shifts. Interestingly, diffusion ordered NMR spectroscopy indicates protons to become more mobile than the constituting acid and base molecules, consistent with Grotthuss-like proton transport. To vary the immediate environment of the PILs, we study solutions of the PIL mixtures in aprotic dimethylformamide (DMF) and protic methanol (MeOH) as two limiting cases. We find that [MimH][AcO] exclusively forms electroneutral species in DMF, as evident from the negligible conductivity, while with increasing TFA content the fraction of charged molecular species increases. Although these ionic species give rise to a finite conductivity, demonstrating the presence of mobile ions, a large fraction of these ions is bound in [MimH][TFA] ion-pairs, as detected in the DR spectra. Yet, temperature dependent 1H-NMR linewidths of the acidic proton signals suggest proton exchange to occur on the NMR timescale, presumably between [MimH]+ and [Mim]. Conversely, in MeOH we find no evidence for the presence of ion-pairs, while the high conductivity of these solutions shows that MeOH can efficiently solvate ions and may participate in proton transfer. Together, our results highlight the sensitivity of protonation equilibria to the immediate environment and of proton dynamics to these protonation equilibria. Our results show that tuning these equilibria using PIL mixtures can provide routes to tailor the contribution of proton transport to charge transport in PILs.

Excited-State Dynamics in 4-[4'(Dimethylamino)styryl]pyridine, a Photobase: Role of Photoinitiated Proton-Coupled Electron Transfer.

The photoinitiated proton-coupled electron transfer (PCET) process in photoacid-based adducts is predominantly governed by the evolution of the electron-proton transfer state. However, such a process is underexplored in the case of photobases as the excited states evolve through multiple competitive channels. Here, we elucidate the excited-state dynamics of a photobase, 4-[4'-(dimethylamino)styryl]pyridine (DMASP), in the presence of hexafluoroisopropanol (HFIP) that enables PCET. Transient absorption measurements show the evolution of a protonated species in the excited state with a time constant of ∼2.5 ps. Fluorescence upconversion measurements reveal the signatures of an emissive intramolecular charge transfer state and a protonated state. The role of such states is further confirmed by time-resolved measurements in the presence of trifluoroacetic acid and computational analysis. Furthermore, the proton-abstraction dynamics of DMASP is analyzed in bulk methanol and butanol solvents. The extent of proton abstraction by DMASP is found to be higher in the presence of HFIP when compared with the normal alcohols over a time period of a few picoseconds.

Development of an Iron(II) Complex Exhibiting Thermal- and Photoinduced Double Proton-Transfer-Coupled Spin Transition in a Short Hydrogen Bond.

Multiple proton transfer (PT) controllable by external stimuli plays a crucial role in fundamental chemistry, biological activity, and material science. However, in crystalline systems, controlling multiple PT, which results in a distinct protonation state, remains challenging. In this study, we developed a novel tridentate ligand and iron(II) complex with a short hydrogen bond (HB) that exhibits a PT-coupled spin transition (PCST). Single-crystal X-ray and neutron diffraction measurements revealed that the positions of the two protons in the complex can be controlled by temperature and photoirradiation based on the thermal- and photoinduced PCST. The obtained results suggest that designing molecules that form short HBs is a promising approach for developing multiple PT systems in crystals.

Quantum Computer Simulation of Protein Protonation.

In attempts to simulate the protonation of proteins, a major challenge is that the number of protonation states grows rapidly as a function (2N) of the number of protonation sites (N). Expression on the free energy of the protonation state as an N-site Ising model ─ using an empirical Generalized-Born model ─ allows a quantum computer to efficiently determine the important states at a given pH value and subsequently reconstruct the pH titration process at all sites. Compared with the exact results painstakingly obtained with classical computers, the results obtained using quantum computers show good agreement for staphylococcal nuclease and excellent agreement for α-lactalbumin. This work illustrates the effectiveness of quantum computers in sampling important physical states, which may be useful in attacking challenging biomolecular problems.

Coexistence of Cationic and Anionic Phosphate Moieties in Solids: Unusual but Not Impossible.

Phosphoric acid is commonly known either as a neutral molecule or as an anion (phosphate). We theoretically confirm by ab initio molecular dynamics simulations (AIMD) that a cationic form H4PO4+ coexists with the anionic form H2PO4- in the same salt. This paradoxical situation is achieved by partial substitution of Cs+ by H4PO4+ in CsH2PO4. Thus, HnPO4 acts simultaneously as both the positive and the negative ion of the salt. We analyze the dynamical protonation pattern within the unusual hydrogen bond network that is established between the ions. Our AIMD simulations show that a conventional assignment of protonation states of the phosphate groups is not meaningful. Instead, a better description of the protonation situation is achieved by an efficiently fractional assignment of the strongly hydrogen-bonded protons to both its nearest and next-nearest oxygen neighbors.

Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B6-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.

Thermodynamically consistent determination of free energies and rates in kinetic cycle models

Kinetic and thermodynamic models of biological systems are commonly used to connect microscopic features to system function in a bottom-up multiscale approach. The parameters of such models—free energy differences for equilibrium properties and in general rates for equilibrium and out-of-equilibrium observables—have to be measured by different experiments or calculated from multiple computer simulations. All such parameters necessarily come with uncertainties so that when they are naively combined in a full model of the process of interest, they will generally violate fundamental statistical mechanical equalities, namely detailed balance and an equality of forward/backward rate products in cycles due to Hill. If left uncorrected, such models can produce arbitrary outputs that are physically inconsistent. Here, we develop a maximum likelihood approach (named multibind) based on the so-called potential graph to combine kinetic or thermodynamic measurements to yield state-resolved models that are thermodynamically consistent while being most consistent with the provided data and their uncertainties. We demonstrate the approach with two theoretical models, a generic two-proton binding site and a simplified model of a sodium/proton antiporter. We also describe an algorithm to use the multibind approach to solve the inverse problem of determining microscopic quantities from macroscopic measurements and, as an example, we predict the microscopic pKa values and protonation states of a small organic molecule from 1D NMR data. The multibind approach is applicable to any thermodynamic or kinetic model that describes a system as transitions between well-defined states with associated free energy differences or rates between these states. A Python package multibind, which implements the approach described here, is made publicly available under the MIT Open Source license.

Ruthenium terpyridine Phenol-Substituent supports PCET and semiquinone-like behavior

Ligands play a central role in dictating the electronic properties of metal complexes to which they are coordinated. A fundamental understanding of changes in ligand properties can be used as design principles for more efficient catalysts. Designing ligands that have multiple protonation states that will change the properties of the coordination complex would be useful as potential ways of controlling catalysis, for example, as an on/off switch where one redox state exists below thermodynamic potential and another exists above. Thus, phenol moieties built into strongly coordinating ligands, like that of tpyPhOH (4′-(4-hydroxyphenyl)-2,2′:6′,2′'-terpyridine) may provide such a handle. Herein, we report the electrochemical and spectral characterization, and the crystallographic and computational analysis of two ruthenium analogs: [Ru(tpy)(tpyPhOH)](PF6)2 and [Ru(tpyPhOH)2](PF6)2 (tpy = 2,2′:6′,2′'-terpyridine). Cyclic voltammetry and differential pulse voltammetry indicate that two redox events occur, one of which is pH independent and we hypothesize that these follow an electrochemical-chemical-electrochemical (ECE) mechanism. XRD results of the ruthenium complexes’ protonated forms are generally consistent with expected bond lengths and angles and are in agreement with computational modeling. The properties are compared to a previously reported analog that contains the –OH group directly connected to terpyridine, [Ru(tpyOH)2](PF6)2, where tpyOH is 4′-hydroxy-2,2′:6′,2′'-terpyridine, with some intriguing differences. Overall, these data indicate that the phenyl-substituent decouples the phenol such that it behaves both as an electron withdrawing substituent and a location for a ligand centered oxidation event to occur.

Variations of the canonical triad of IsPETase: In silico insights with molecular dynamics simulation

Plastic waste causes severe environmental problems worldwide, and biological recycling is an eco-friendly method to deal with it. The discovery of IsPETase has attracted interest in the scientific community. Its mesophilic nature is an exciting characteristic for new perspectives into PET degradation research. Here, we used a molecular dynamics approach and discussed the possibility of variations for the canonical catalytic triad at pH 7 and 300 K. We prepared three variants and performed a comparative molecular docking protocol of BHET in these three and the wild type. We performed molecular dynamics focusing on the nucleophilic distance and the protonation states of the residues chosen on our mutations. In all simulations, the ligand remained bound to the active site. The total binding energy on the variants diminished but always remained favorable for interaction. Upon analysis of the interaction contribution of each residue, the simulations with the wild type and D206E presented a more stable bond with basically the same binding energy pattern. However, the lysine variants showed a reduction in the unfavorable contributions – while D206E/H237K did not present any unfavorability whatsoever. The new charge distribution and side chains conformation for both lysine variants kept the nucleophilic distance while pushing the ligand away from the oxyanion hole. The single mutants studied here did not show promising results. However, the peculiar behavior of D206E/H237K provokes curiosity about what consequences may arise with empirical data. We believe the discussion here may benefit future efforts regarding PET degradation efficiency at ambient conditions.

Polymorphic amyloid nanostructures of hormone peptides involved in glucose homeostasis display reversible amyloid formation

A large group of hormones are stored as amyloid fibrils in acidic secretion vesicles before they are released into the bloodstream and readopt their functional state. Here, we identify an evolutionarily conserved hexapeptide sequence as the major aggregation-prone region (APR) of gastrointestinal peptides of the glucagon family: xFxxWL. We determine nine polymorphic crystal structures of the APR segments of glucagon-like peptides 1 and 2, and exendin and its derivatives. We follow amyloid formation by CD, FTIR, ThT assays, and AFM. We propose that the pH-dependent changes of the protonation states of glutamate/aspartate residues of APRs initiate switching between the amyloid and the folded, monomeric forms of the hormones. We find that pH sensitivity diminishes in the absence of acidic gatekeepers and amyloid formation progresses over a broad pH range. Our results highlight the dual role of short aggregation core motifs in reversible amyloid formation and receptor binding.