Protonation State Research Articles

SwissParam 2023: A Modern Web-Based Tool for Efficient Small Molecule Parametrization.

Most steps of drug discovery are now routinely supported and accelerated by computer-aided drug design tools. Among them, structure-based approaches use the three-dimensional structure of the targeted biomacromolecule as a major source of information. When it comes to calculating the interactions of small molecules with proteins using the equations of molecular mechanics, topologies, atom typing, and force field parameters are required. However, generating parameters for small molecules remains challenging due to the large number of existing chemical groups. The SwissParam web tool was first released in 2011 with the aim of generating parameters and topologies for small molecules based on the Merck molecular force field (MMFF) while being compatible with the CHARMM22/27 force field. Here, we present an updated version of SwissParam, providing various new features, including the possibility to setup covalent ligands. Molecules can now be imported from different file formats or via a molecular sketcher. The MMFF-based approach has been updated to provide parameters and topologies compatible with the CHARMM36 force field. An option was added to generate small molecule parametrizations following the CHARMM General Force Field via the multipurpose atom-typer for CHARMM (MATCH) approach. Additionally, SwissParam now generates information on probable alternative tautomers and protonation states of the query molecule so that the user can consider all microspecies relevant to its compound. The new version of SwissParam is freely available at www.swissparam.ch and can also be accessed through a newly implemented command-line interface.

Amide isomerization pathways: Electronic and structural background of protonation- and deprotonation-mediated cis-trans interconversions.

The cis-trans isomerization of amide bonds leads to wide range of structural and functional changes in proteins and can easily be the rate-limiting step in folding. The trans isomer is thermodynamically more stable than the cis, nevertheless the cis form can play a role in biopolymers' function. The molecular system of N-methylacetamide · 2H2O is complex enough to reveal energetics of the cis-trans isomerization at coupled cluster single-double and coupled cluster single-double and perturbative triple [CCSD(T)] levels of theory. The cis-trans isomerization cannot be oversimplified by a rotation along ω, since this rotation is coupled with the N-atom pyramidal inversion, requesting the introduction of a second dihedral angle "α." Full f(ω,α) potential energy surfaces of the different amide protonation states, critical points and isomerization reaction paths were determined, and the barriers of the neutral, O-protonated and N-deprotonated amides were found too high to allow cis-trans interconversion at room temperature: ∼85, ∼140, and ∼110 kJ mol-1, respectively. For the N-protonated amide bond, the cis form (ω = 0°) is a maximum rather than a minimum, and each ω state is accessible for less than ∼10 kJ mol-1. Here we outline a cis-trans isomerization pathway with a previously undescribed low energy transition state, which suggests that the proton is transferred from the more favorable O- to the N-protonation site with the aid of nearby water molecules, allowing the trans → cis transition to occur at an energy cost of ≤11.6 kJ mol-1. Our results help to explain why isomerase enzymes operate via protonated amide bonds and how N-protonation of the peptide bond occurs via O-protonation.

Reverse pH-dependent fluorescence protein visualizes pattern of interfacial proton dynamics during hydrogen evolution reaction

Reverse pH-dependent fluorescent protein, including dKeima, is a type of fluorescent protein in which the chromophore protonation state depends inversely on external pH. The dependence is maintained even when immobilized at the metal-solution interface. But, interestingly, its responses to the hydrogen evolution reaction (HER) at the interface are not reversed: HER rises the pH of the solution around the cathode, but, highly active HER induces chromophore deprotonation regardless of the reverse pH dependence, reflecting an interface-specific deprotonation effect by HER. Here, we exploit this phenomenon to perform scanning-less, real-time visualization of interfacial proton dynamics during HER at a wide field of view. By using dKeima, the HER-driven deprotonation effect was well discriminated from the solution pH effect. In the electrodes of composite structures with a catalyst, dKeima visualized keen dependence of the proton depletion pattern on the electrode configuration. In addition, propagations of optical signals were observed, which seemingly reflect long-range proton hopping confined to the metal-solution interface. Thus, reverse pH-dependent fluorescent proteins provide a unique tool for spatiotemporal analysis of interfacial proton dynamics, which is expected to contribute to a better understanding of the HER process and ultimately to the safe and efficient production of molecular hydrogen.

Molecular Dynamics for the Optimal Design of Functionalized Nanodevices to Target Folate Receptors on Tumor Cells.

Atomistic details on the mechanism of targeting activity by biomedical nanodevices of specific receptors are still scarce in the literature, where mostly ligand/receptor pairs are modeled. Here, we use atomistic molecular dynamics (MD) simulations, free energy calculations, and machine learning approaches on the case study of spherical TiO2 nanoparticles (NPs) functionalized with folic acid (FA) as the targeting ligand of the folate receptor (FR). We consider different FA densities on the surface and different anchoring approaches, i.e., direct covalent bonding of FA γ-carboxylate or through polyethylene glycol spacers. By molecular docking, we first identify the lowest energy conformation of one FA inside the FR binding pocket from the X-ray crystal structure, which becomes the starting point of classical MD simulations in a realistic physiological environment. We estimate the binding free energy to be compared with the existing experimental data. Then, we increase complexity and go from the isolated FA to a nanosystem decorated with several FAs. Within the simulation time framework, we confirm the stability of the ligand-receptor interaction, even in the presence of the NP (with or without a spacer), and no significant modification of the protein secondary structure is observed. Our study highlights the crucial role played by the spacer, FA protonation state, and density, which are parameters that can be controlled during the nanodevice preparation step.

Stabilization of a Heme-HNO Model Complex Using a Bulky Bis-Picket Fence Porphyrin and Reactivity Studies with NO.

Nitroxyl, HNO/NO-, the one-electron reduced form of NO, is suggested to take part in distinct signaling pathways in mammals and is also a key intermediate in various heme-catalyzed NOx interconversions in the nitrogen cycle. Cytochrome P450nor (Cyt P450nor) is a heme-containing enzyme that performs NO reduction to N2O in fungal denitrification. The reactive intermediate in this enzyme, termed "Intermediate I", is proposed to be an Fe-NHO/Fe-NHOH type species, but it is difficult to study its electronic structure and exact protonation state due to its instability. Here, we utilize a bulky bis-picket fence porphyrin to obtain the first stable heme-HNO model complex, [Fe(3,5-Me-BAFP)(MI)(NHO)], as a model for Intermediate I, and more generally HNO adducts of heme proteins. Due to the steric hindrance of the bis-picket fence porphyrin, [Fe(3,5-Me-BAFP)(MI)(NHO)] is stable (τ1/2 = 56 min at -30 °C), can be isolated as a solid, and is available for thorough spectroscopic characterization. In particular, we were able to solve a conundrum in the literature and provide the first full vibrational characterization of a heme-HNO complex using IR and nuclear resonance vibrational spectroscopy (NRVS). Reactivity studies of [Fe(3,5-Me-BAFP)(MI)(NHO)] with NO gas show a 91 ± 10% yield for N2O formation, demonstrating that heme-HNO complexes are catalytically competent intermediates for NO reduction to N2O in Cyt P450nor. The implications of these results for the mechanism of Cyt P450nor are further discussed.

Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins.

Red fluorescent proteins (RFPs) represent an increasingly popular class of genetically encodable bioprobes and biomarkers that can advance next-generation breakthroughs across the imaging and life sciences. Since the rational design of RFPs with improved functions or enhanced versatility requires a mechanistic understanding of their working mechanisms, while fluorescence is intrinsically an ultrafast event, a suitable toolset involving steady-state and time-resolved spectroscopic techniques has become powerful in delineating key structural features and dynamic steps which govern irreversible photoconverting or reversible photoswitching RFPs, and large Stokes shift (LSS)RFPs. The pertinent cis-trans isomerization and protonation state change of RFP chromophores in their local environments, involving key residues in protein matrices, lead to rich and complicated spectral features across multiple timescales. In particular, ultrafast excited-state proton transfer in various LSSRFPs showcases the resolving power of wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in mapping a photocycle with crucial knowledge about the red-emitting species. Moreover, recent progress in noncanonical RFPs with a site-specifically modified chromophore provides an appealing route for efficient engineering of redder and brighter RFPs, highly desirable for bioimaging. Such an effective feedback loop involving physical chemists, protein engineers, and biomedical microscopists will enable future successes to expand fundamental knowledge and improve human health.

Accurately Predicting Protein pKa Values Using Nonequilibrium Alchemy.

The stability, solubility, and function of a protein depend on both its net charge and the protonation states of its individual residues. pKa is a measure of the tendency for a given residue to (de)protonate at a specific pH. Although pKa values can be resolved experimentally, theory and computation provide a compelling alternative. To this end, we assess the applicability of a nonequilibrium (NEQ) alchemical free energy method to the problem of pKa prediction. On a data set of 144 residues that span 13 proteins, we report an average unsigned error of 0.77 ± 0.09, 0.69 ± 0.09, and 0.52 ± 0.04 pK for aspartate, glutamate, and lysine, respectively. This is comparable to current state-of-the-art predictors and the accuracy recently reached using free energy perturbation methods (e.g., FEP+). Moreover, we demonstrate that our open-source, pmx-based approach can accurately resolve the pKa values of coupled residues and observe a substantial performance disparity associated with the lysine partial charges in Amber14SB/Amber99SB*-ILDN, for which an underused fix already exists.

Protonation State and Proton Conductivity Mechanism in Supramolecular Three-Armed Histamine-Based Derivative Self-Assembly

Protonation State and Proton Conductivity Mechanism in Supramolecular Three-Armed Histamine-Based Derivative Self-Assembly

Deciphering the Catalytic Mechanism of Virginiamycin B Lyase with Multiscale Methods and Molecular Dynamics Simulations.

Due to the emergence of antibiotic resistance, the need to explore novel antibiotics and/or novel strategies to counter antibiotic resistance is of utmost importance. In this work, we explored the molecular and mechanistic details of the degradation of a streptogramin B antibiotic by virginiamycin B (Vgb) lyase of Staphylococcus aureus using classical molecular dynamics simulations and multiscale quantum mechanics/molecular mechanics methods. Our results were in line with available experimental kinetic information. Although we were able to identify a stepwise mechanism, in the wild-type enzyme, the intermediate is short-lived, showing a small barrier to decay to the product state. The impact of point mutations on the reaction was also assessed, showing not only the importance of active site residues to the reaction catalyzed by Vgb lyase but also of near positive and negative residues surrounding the active site. Using molecular dynamics simulations, we also predicted the most likely protonation state of the 3-hydroxypicolinic moiety of the antibiotic and the impact of mutants on antibiotic binding. All this information will expand our understanding of linearization reactions of cyclic antibiotics, which are crucial for the development of novel strategies that aim to tackle antibiotic resistance.

In Silico, In Vitro and In Vivo Assessment of Acetylcholinesterase Inhibitory Activity of Theobromine Derivatives Containing an Arylpiperazine Fragment

Background: In the current Alzheimer’s disease therapy as the preferred treatment are applied acetylcholinesterase inhibitors. Aiming to identify the active pharmacophores necessary for increased acetylcholinesterase inhibitory activity, some docking studies have been applied. Methods: In silico docking evaluation of the binding modes, identification of acetylcholinesterase inhibitory activity in vitro through Ellman’s test and ITC protocol, and the in vivo effect. PAMPA evaluation of the GIT and BBB permeability. Results: In the present study, two series previously synthesized in our laboratory, arylpiperazine derivatives of theobromine were docked into the rhAChE active sites. Ellman’s test outlined molecules LA1 and LA7 as the most active, with IC50 of 0.708 and 0.299 μM, respectively. In the acute toxicity test, LA7 given intraperitoneally in mice showed moderate toxicity with LD50 of 87.5 mg/kg. The new compound, administered i.p. for 12 days at doses 2 mg/kg/day and 4 mg/kg/day, respectively, showed a pronounced acetylcholinesterase inhibitory activity in vivo. Conclusion: The corresponding binding modes were identified, where the docking pose for the studied molecules depends on the protonated state of the nitrogen atom of the piperazine moiety. In the best scored pose for LA7, the xanthine moiety is bound into the catalytic active site (CAS) of acetylcholinesterase, while the arylpiperazine fragment is placed into the peripheral binding site (PAS). For the evaluated selected structures, good permeability through the GIT and BBB assessed by PAMPA was also determined.

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.