Protonation State Research Articles

Computational Analysis of Histamine Protonation Effects on H1R Binding.

Despite numerous studies investigating histamine and its receptors, the impact of histamine protonation states on binding to the histamine H1-receptor (H1R) has remained elusive. Therefore, we assessed the influence of different histamine tautomers (τ-tautomer, π-tautomer) and charge states (mono- vs. dicationic) on the interaction with the ternary histamine-H1R-Gq complex. In atomistic molecular dynamics simulations, the τ-tautomer formed stable interactions with the receptor, while the π-tautomer induced a rotation of the histamine ring by 180° and formed only weaker hydrogen bonding interactions. This suggests that the τ-tautomer is more relevant for stabilization of the active ternary histamine-H1R-Gq complex. In addition to the two monocationic tautomers, the binding of dicationic histamine was investigated, whose interaction with the H1R had been observed in a previous experimental study. Our simulations showed that the dication is less compatible with the ternary histamine-H1R-Gq complex and rather induces an inactive conformation in the absence of the Gq protein. Our data thus indicate that the charge state of histamine critically affects its interactions with the H1R. Ultimately these findings might have implications for the future development of new ligands that stabilize distinct H1R activation states.

Combined Structural and Computational Study of the mRubyFT Fluorescent Timer Locked in Its Blue Form.

The mRubyFT is a monomeric genetically encoded fluorescent timer based on the mRuby2 fluorescent protein, which is characterized by the complete maturation of the blue form with the subsequent conversion to the red one. It has higher brightness in mammalian cells and higher photostability compared with other fluorescent timers. A high-resolution structure is a known characteristic of the mRubyFT with the red form chromophore, but structural details of its blue form remain obscure. In order to obtain insight into this, we obtained an S148I variant of the mRubyFT (mRubyFTS148I) with the blocked over time blue form of the chromophore. X-ray data at a 1.8 Å resolution allowed us to propose a chromophore conformation and its interactions with the neighboring residues. The imidazolidinone moiety of the chromophore is completely matured, being a conjugated π-system. The methine bridge is not oxidized in the blue form bringing flexibility to the phenolic moiety that manifests itself in poor electron density. Integration of these data with the results of molecular dynamic simulation disclosed that the OH group of the phenolic moiety forms a hydrogen bond with the side chain of the T163 residue. A detailed comparison of mRubyFTS148I with other available structures of the blue form of fluorescent proteins, Blue102 and mTagBFP, revealed a number of characteristic differences. Molecular dynamic simulations with the combined quantum mechanic/molecular mechanic potentials demonstrated that the blue form exists in two protonation states, anion and zwitterion, both sharing enolate tautomeric forms of the C=C-O- fragment. These two forms have similar excitation energies, as evaluated by calculations. Finally, excited state molecular dynamic simulations showed that excitation of the chromophore in both protonation states leads to the same anionic fluorescent state. The data obtained shed light on the structural features and spectral properties of the blue form of the mRubyFT timer.

Prospects for a lattice calculation of the rare decay Σ+ → pℓ+ℓ−

We present a strategy for calculating the rare decay of a Σ+(uus) baryon to a proton (uud) and di-lepton pair using lattice QCD. To determine this observable one needs to numerically evaluate baryonic two-, three-, and four-point correlation functions related to the target process. In particular, the four-point function arises from the insertion of incoming and outgoing baryons, together with a weak Hamiltonian mediating the s → d transition and an electromagnetic current creating the outgoing leptons. As is described in previous work in other contexts, this four-point function has a highly non-trivial relation to the physical observable, due to proton and proton-pion intermediate states. These lead to growing Euclidean time dependence and, in the case of the two-particle intermediate states, to power-like volume effects. We discuss how to treat these issues in the context of the Σ+ → pℓ+ℓ− decay and, in particular, detail the relation between the finite-volume estimator and the physical, complex-valued amplitude. In doing so, we also make connections between various approaches already described in the literature.

Epik: pKa and Protonation State Prediction through Machine Learning

Epik version 7 is a software program that uses machine learning for predicting the pKa values and protonation state distribution of complex, druglike molecules. Using an ensemble of atomic graph convolutional neural networks (GCNNs) trained on over 42,000 pKa values across broad chemical space from both experimental and computed origins, the model predicts pKa values with 0.42 and 0.72 pKa unit median absolute and root mean square errors, respectively, across seven test sets. Epik version 7 also generates protonation states and recovers 95% of the most populated protonation states compared to previous versions. Requiring on average only 47 ms per ligand, Epik version 7 is rapid and accurate enough to evaluate protonation states for crucial molecules and prepare ultra-large libraries of compounds to explore vast regions of chemical space. The simplicity and time required for the training allow for the generation of highly accurate models customized to a program's specific chemistry.

Elucidating the Mechanism for the Reaction of o-Phthalaldehyde with Primary Amines in the Presence of Thiols.

The use of o-phthalaldehyde (OPA) in combination with a thiol reagent is a common method for detecting primary amines in amino acids, peptides, and proteins. Despite its widespread use, the exact reaction mechanism has been debated since the 1980s. Here, we measure the kinetics of the reaction between OPA, alanine, and a dithiol (1,4-dithiolthreitol, DTT) as a function of pH and reagent concentration. Using these new measurements and accompanying kinetic models, we find evidence that the pH dependence of the kinetics arises from both the protonation states of alanine and DTT, the hydration state of OPA, and the unproductive equilibrium with DTT, all of which are pH-dependent. These results support the mechanism originally proposed by Sternson [Rational design and evaluation of improved o-phthalaldehyde-like fluorogenic reagents. Anal. Biochem. 1985, 144, 233-246] and Wong [Reaction of o-phthalaldehyde with alanine and thiols: kinetics and mechanism. J. Am. Chem. Soc. 1985, 107, 6421-6422], in which the primary amine first reacts with OPA, followed by a reaction with the thiol to form the fluorescent isoindole product.

The molecular basis of the antidepressant action of the magic mushroom extract, psilocin

Magic mushrooms, and their extract psilocybin, are well-known for their psychedelic properties and recreational use. Psilocin, the bio-active form of psilocybin, can potentially treat various psychiatric diseases. Psilocin putatively exerts its psychedelic effect as an agonist to the serotonin 2A receptor (5-HT2AR), which is also the receptor for the neurological hormone serotonin. The two key chemical differences between the two molecules are first, that the primary amine in serotonin is replaced with a tertiary amine in psilocin, and second, the hydroxyl group is substituted differently on the aromatic ring. Here, we find that psilocin can bind to 5-HT2AR with an affinity higher than serotonin, and provide the molecular logic behind the higher binding affinity of psilocin using extensive molecular dynamics simulations and free energy calculations. The binding free energy of psilocin is dependent upon the protonation states of the ligands, as well as that of the key residue in the binding site: Aspartate 155. We find that the tertiary amine of psilocin, and not the altered substitution of the hydroxyl group in the ring is responsible for the increased affinity of psilocin. We propose design rules for effective antidepressants based on molecular insights from our simulations.

Exploring the Macroscopic Properties of Humic Substances Using Modeling and Molecular Simulations

Soil organic matter (SOM) is composed of a complex and heterogeneous mixture of organic compounds, which poses a challenge in understanding it on an atomistic level. Based on the progress of molecular dynamics simulations and our efforts to create molecular systems that resemble SOM, in this work, we expanded our knowledge of SOM through the use of humic substances (HSs). Specifically, we studied the standardized samples of HS of the International Humic Substances Society (IHSS). This society provided the elemental and organic composition used as input parameters for our Vienna Soil Organic Matter Modeler 2 (VSOMM2). We modeled and simulated different HS samples from various sources, including soil, peat, leonardite, and blackwater river. In order to compare between different HS, we reduced the organic composition information to two principal components, which are associated principally with the amount of carboxyl and aromatic groups in the HS, denominated as PCacid and PCarom, respectively. We performed a plethora of analyses to characterize the structure and dynamics of the systems, including the total potential energy, density, diffusion, preferential solvation, hydrogen bonds, and salt bridges. In general terms, at the water content value of 0.2, we observed that most properties depend on the carboxyl group protonation state. The Coulombic interactions from this ionic specie and the interaction with cations determine the overall behavior of the studied systems. Furthermore, the type of cations and the pH influence those properties. This study exemplifies the importance of molecular dynamics to explain macroscopic properties from the structure and dynamics of the molecules modeled, such as the interaction network, i.e., hydrogen bonds or salt bridges of molecules presented in the system and their mobility.

Unusual pKa Values Mediate the Self-Assembly of Spider Dragline Silk Proteins.

Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental pKa values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (∼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with pKas above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical pKa values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.

Understanding the Electronic Structure Basis for N2 Binding to FeMoco: A Systematic Quantum Mechanics/Molecular Mechanics Investigation.

The FeMo cofactor (FeMoco) of Mo nitrogenase is responsible for reducing dinitrogen to ammonia, but it requires the addition of 3-4 e-/H+ pairs before N2 even binds. A binding site at the Fe2/Fe3/Fe6/Fe7 face of the cofactor has long been suggested based on mutation studies, with Fe2 or Fe6 nowadays being primarily discussed as possibilities. However, the nature of N2 binding to the cofactor is enigmatic as the metal ions are coordinatively saturated in the resting state with no obvious binding site. Furthermore, the cofactor consists of high-spin Fe(II)/Fe(III) ions (antiferromagnetically coupled but also mixed-valence delocalized), which are not known to bind N2. This suggests that an Fe binding site with a different molecular and electronic structure than the resting state must be responsible for the experimentally known N2 binding in the E4 state of FeMoco. We have systematically studied N2 binding to Fe2 and Fe6 sites of FeMoco at the broken-symmetry QM/MM level as a function of the redox-, spin-, and protonation state of the cofactor. The local and global electronic structure changes to the cofactor taking place during redox events, protonation, Fe-S cleavage, hydride formation, and N2 coordination are systematically analyzed. Localized orbital and quasi-restricted orbital analysis via diamagnetic substitution is used to get insights into the local single Fe ion electronic structure in various states of FeMoco. A few factors emerge as essential to N2 binding in the calculations: spin-pairing of dxz and dyz orbitals of the N2-binding Fe ion, a coordination change at the N2-binding Fe ion aided by a hemilabile protonated sulfur, and finally hydride ligation. The results show that N2 binding to E0, E1, and E2 models is generally unfavorable, likely due to the high-energy cost of stabilizing the necessary spin-paired electronic structure of the N2-binding Fe ion in a ligand environment that clearly favors high-spin states. The results for models of E4, however, suggest a feasible model for why N2 binding occurs experimentally in the E4 state. E4 models with two bridging hydrides between Fe2 and Fe6 show much more favorable N2 binding than other models. When two hydrides coordinate to the same Fe ion, an increased ligand-field splitting due to octahedral coordination at either Fe2 or Fe6 is found. This altered ligand field makes it easier for the Fe ion to acquire a spin-paired electronic structure with doubly occupied dxz and dyz orbitals that backbond to a terminal N2 ligand. Within this model for N2 binding, both Fe2 and Fe6 emerge as possible binding site scenarios. Due to steric effects involving the His195 residue, affecting both the N2 ligand and the terminal SH- group, distinguishing between Fe2 and Fe6 is difficult; furthermore, the binding depends on the protonation state of His195.

Protonation states of Asp residues in the human Nudix hydrolase MTH1 contribute to its broad substrate recognition.

Human MutT homolog 1 (MTH1), also known as Nudix-type motif 1 (NUDT1), hydrolyzes 8-oxo-dGTP and 2-oxo-dATP with broad substrate recognition and has attracted attention in anticancer therapeutics. Previous studies on MTH1 have proposed that the exchange of the protonation state between Asp119 and Asp120 is essential for the broad substrate recognition of MTH1. To understand the relationship between protonation states and substrate binding, we determined the crystal structures of MTH1 at pH 7.7-9.7. With increasing pH, MTH1 gradually loses its substrate-binding ability, indicating that Asp119 is deprotonated at pH 8.0-9.1 in 8-oxo-dGTP recognition and Asp120 is deprotonated at pH 8.6-9.7 in 2-oxo-dATP recognition. These results confirm that MTH1 recognizes 8-oxo-dGTP and 2-oxo-dATP by exchanging the protonation state between Asp119 and Asp120 with higher pKa .

Modern semiempirical electronic structure methods and machine learning potentials for drug discovery: Conformers, tautomers, and protonation states.

Modern semiempirical electronic structure methods have considerable promise in drug discovery as universal "force fields" that can reliably model biological and drug-like molecules, including alternative tautomers and protonation states. Herein, we compare the performance of several neglect of diatomic differential overlap-based semiempirical (MNDO/d, AM1, PM6, PM6-D3H4X, PM7, and ODM2), density-functional tight-binding based (DFTB3, DFTB/ChIMES, GFN1-xTB, and GFN2-xTB) models with pure machine learning potentials (ANI-1x and ANI-2x) and hybrid quantum mechanical/machine learning potentials (AIQM1 and QDπ) for a wide range of data computed at a consistent ωB97X/6-31G* level of theory (as in the ANI-1x database). This data includes conformational energies, intermolecular interactions, tautomers, and protonation states. Additional comparisons are made to a set of natural and synthetic nucleic acids from the artificially expanded genetic information system that has important implications for the design of new biotechnology and therapeutics. Finally, we examine the acid/base chemistry relevant for RNA cleavage reactions catalyzed by small nucleolytic ribozymes, DNAzymes, and ribonucleases. Overall, the hybrid quantum mechanical/machine learning potentials appear to be the most robust for these datasets, and the recently developed QDπ model performs exceptionally well, having especially high accuracy for tautomers and protonation states relevant to drug discovery.

Ruthenium Complexes with Protic Ligands: Influence of the Position of OH Groups and π Expansion on Luminescence and Photocytotoxicity

Protic ruthenium complexes using the dihydroxybipyridine (dhbp) ligand combined with a spectator ligand (N,N = bpy, phen, dop, Bphen) have been studied for their potential activity vs. cancer cells and their photophysical luminescent properties. These complexes vary in the extent of π expansion and the use of proximal (6,6'-dhbp) or distal (4,4'-dhbp) hydroxy groups. Eight complexes are studied herein as the acidic (OH bearing) form, [(N,N)2Ru(n,n'-dhbp)]Cl2, or as the doubly deprotonated (O- bearing) form. Thus, the presence of these two protonation states gives 16 complexes that have been isolated and studied. Complex 7A, [(dop)2Ru(4,4'-dhbp)]Cl2, has been recently synthesized and characterized spectroscopically and by X-ray crystallography. The deprotonated forms of three complexes are also reported herein for the first time. The other complexes studied have been synthesized previously. Three complexes are light-activated and exhibit photocytotoxicity. The log(Do/w) values of the complexes are used herein to correlate photocytotoxicity with improved cellular uptake. For Ru complexes 1-4 bearing the 6,6'-dhbp ligand, photoluminescence studies (all in deaerated acetonitrile) have revealed that steric strain leads to photodissociation which tends to reduce photoluminescent lifetimes and quantum yields in both protonation states. For Ru complexes 5-8 bearing the 4,4'-dhbp ligand, the deprotonated Ru complexes (5B-8B) have low photoluminescent lifetimes and quantum yields due to quenching that is proposed to involve the 3LLCT excited state and charge transfer from the [O2-bpy]2- ligand to the N,N spectator ligand. The protonated OH bearing 4,4'-dhbp Ru complexes (5A-8A) have long luminescence lifetimes which increase with increasing π expansion on the N,N spectator ligand. The Bphen complex, 8A, has the longest lifetime of the series at 3.45 μs and a photoluminescence quantum yield of 18.7%. This Ru complex also exhibits the best photocytotoxicity of the series. A long luminescence lifetime is correlated with greater singlet oxygen quantum yields because the triplet excited state is presumably long-lived enough to interact with 3O2 to yield 1O2.

Room-Temperature One-Pot Synthesis of pH-Responsive Pyridine-Functionalized Carbon Surfaces.

Carbon surfaces (glassy carbon, graphite, and boron-doped diamond) were functionalized with layers composed of linked pyridinium and pyridine moieties using simple electrochemical reduction of trifluoroacetylpyridinium. The pyridinium species was generated in situ in solution by the reaction of trifluoroacetic anhydride and pyridine precursors and underwent electrochemical reduction at -1.97 V vs Fc/Fc+, as determined by cyclic voltammetry. The pyridine/pyridinium films were electrodeposited at room temperature, on a timescale of minutes, and were characterized using X-ray photoelectron spectroscopy. The as-prepared films have a net positive charge in aqueous solution at pH 9 and below due to the pyridinium content, confirmed by the electrochemical response of differently charged redox molecules at the functionalized surfaces. The positive charge can be enhanced further through protonation of the neutral pyridine component by controlling the solution pH. Moreover, the nitrogen-acetyl bond can be cleaved through base treatment to purposefully increase the neutral pyridine proportion of the film. This results in a surface that can be "switched" from functionally near neutral to a positive charge by treatment in basic and acidic solutions, respectively, through manipulation of the protonation state of the pyridine. The functionalization process demonstrated here is readily achievable at a fast timescale at room temperature and hence can allow for rapid screening of surface properties. Such functionalized surfaces present a means to test in isolation the specific catalytic performance of pyridinic groups toward key processes such as oxygen and CO2 reduction.

Identification of the protonation and oxidation states of the oxygen-evolving complex in the low-dose X-ray crystal structure of photosystem II.

In photosystem II (PSII), the O3 and O4 sites of the Mn4CaO5 cluster form hydrogen bonds with D1-His337 and a water molecule (W539), respectively. The low-dose X-ray structure shows that these hydrogen bond distances differ between the two homogeneous monomer units (A and B) [Tanaka etal., J. Am Chem. Soc. 2017, 139, 1718]. We investigated the origin of the differences using a quantum mechanical/molecular mechanical (QM/MM) approach. QM/MM calculations show that the short O4-OW539 hydrogen bond (~2.5 Å) of the B monomer is reproduced when O4 is protonated in the S1 state. The short O3-NεHis337 hydrogen bond of the A monomer is due to the formation of a low-barrier hydrogen bond between O3 and doubly-protonated D1-His337 in the overreduced states (S-1 or S-2). It seems plausible that the oxidation state differs between the two monomer units in the crystal.

The Mechanism of the Channel Opening in Channelrhodopsin-2: A Molecular Dynamics Simulation.

Channelrhodopsin-2 (ChR2) has been one of the most important objects in the study of optogenetics. The retinal chromophore molecule absorbs photons and undergoes an isomerization reaction, which triggers the photocycle, resulting in a series of conformational changes. In this study, a series of intermediate structures (including D470, P500, P390-early, P390-late, and P520 states) of ChR2 in the photocycle were modeled, and molecular dynamics (MD) simulations were performed to elucidate the mechanism of ion channel opening of ChR2. The maximum absorption wavelength of these intermediates calculated by time-dependent density function theory (TD-DFT) is in general agreement with the experimental values, the distribution of water density gradually increases in the process of photocycle, and the radius of the ion channel is larger than 6 Å. All these results indicate that our structural models of the intermediates are reasonable. The evolution of protonation state of E90 during the photocycle is explained. E90 will deprotonate when the P390-early transforms into P390-late, in which the two conformations of P390-early and P390-late obtained from the simulations are consistent with the experimental descriptions. To validate the conductive P520 state, the potential mean force (PMF) of Na+ ions passing through the P520 intermediate was calculated by using steered molecular dynamics (SMD) simulation combined with umbrella sampling. The result shows that the Na+ ions passing through the channel with a very low energy barrier, especially in the central gate, is almost barrierless. This indicates that the channel is open in the P520 state.

Corrosion inhibition of mild steel in acidic media using new oxo-pyrimidine derivatives: Experimental and theoretical insights

Interesting results have been found for new compounds derived from oxo-pyrimidine to protect of mild steel (MS) in 1 M hydrochloric acid. These the compounds are 1-(5-(4-Methoxy-benzoyl)-4-(4‑methoxy-phenyl)-2-oxo-2H-pyrimidin-1-yl]-(4‑methoxy-phenyl)-urea (MMOM) and 1-(5-(4‑methoxy-benzoyl)-4-(4‑methoxy-phenyl)-2-oxo-2H-pyrimidin-1-yl)-3-(4-methlyphenyl)-thiourea (MMOPM). In this study, the impact of immersion time on inhibitor effectiveness was also investigated. Both substances function as mixed-type inhibitors, according to the electrochemical data. At 5 × 10−4 M and a 72-hour immersion duration, MMOM is more effective than MMOPM (98.42% vs. 94.49%). The Langmuir isotherm system provided the best match for both compounds, with chemisorption as the kind of adsorption. According to the findings of surface characterisation, both chemicals may be adsorbed on mild steel surfaces to reduce corrosion. Inhibitor simulations using density functional theory revealed that the protonated state is more reactive than the neutral state and coincides with experimental findings. The outcomes demonstrated that both compounds may be utilised as new mild steel corrosion inhibitors in harsh conditions and long-term immersion. The theoretical study, based on quantum chemical calculations of the compounds, performed by the DFT/BP86 method with a 6–311G(d,p) basis set by using Gaussian 09, Revision A.02 program, were also included to support experimental results. The various quantum chemical parameters such as EHOMO, ELUMO, chemical hardness and chemical softness, electronegativity of the investigated molecules were calculated, and their inhibition efficiency were discussed. The outcomes demonstrated that both compounds may be utilised as new mild steel corrosion inhibitors in harsh conditions and long-term immersion.

Dissipative Systems Driven by the Decarboxylation of Activated Carboxylic Acids.

ConspectusThe achievement of artificial systems capable of being maintained in out-of-equilibrium states featuring functional properties is a main goal of current chemical research. Absorption of electromagnetic radiation or consumption of a chemical species (a "chemical fuel") are the two strategies typically employed to reach such out-of-equilibrium states, which have to persist as long as one of the above stimuli is present. For this reason such systems are often referred to as "dissipative systems". In the simplest scheme, the dissipative system is initially found in a resting, equilibrium state. The addition of a chemical fuel causes the system to shift to an out-of-equilibrium state. When the fuel is exhausted, the system reverts to the initial, equilibrium state. Thus, from a mechanistic standpoint, the dissipative system turns out to be a catalyst for the fuel consumption. It has to be noted that, although very simple, this scheme implies the chance to temporally control the dissipative system. In principle, modulating the nature and/or the amount of the chemical fuel added, one can have full control of the time spent by the system in the out-of-equilibrium state.In 2016, we found that 2-cyano-2-phenylpropanoic acid (1a), whose decarboxylation proceeds smoothly under mild basic conditions, could be used as a chemical fuel to drive the back and forth motion of a catenane-based molecular switch. The acid donates a proton to the catenane that passes from the neutral state A to the transient protonated state B. Decarboxylation of the resulting carboxylate (1acb), generates a carbanion, which, being a strong base, retakes the proton from the protonated catenane that, consequently, returns to the initial state A. The larger the amount of the added fuel, the longer the time spent by the catenane in the transient, out-of-equilibrium state. Since then, acid 1a and other activated carboxylic acids (ACAs) have been used to drive the operation of a large number of dissipative systems based on the acid-base reaction, from molecular machines to host-guest systems, from catalysts to smart materials, and so on. This Account illustrates such systems with the purpose to show the wide applicability of ACAs as chemical fuels. This generality is due to the simplicity of the idea underlying the operation principle of ACAs, which always translates into simple experimental requirements.

ATP binding by an F1Fo ATP synthase ε subunit is pH dependent, suggesting a diversity of ε subunit functional regulation in bacteria.

It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F1Fo ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F1Fo ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F1Fo ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside the ATP/Mg2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F1Fo ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by second-shell residues thus the function of the ε subunit changes with growth conditions.

Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA

Zinc finger proteins specifically recognize DNA sequences and, therefore, play a crucial role in living organisms. In this study the Zn(II)-, and DNA-binding of 1MEY#, an artificial zinc finger protein consisting of three finger units was characterized by multiple methods. Fluorimetric, circular dichroism and isothermal calorimetric titrations were applied to determine the accurate stability constant of a zinc finger protein. Assuming that all three zinc finger subunits behave identically, the obtained thermodynamic data for the Zn(II) binding were ΔHbinding site = − (23.5 − 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and logβ’pH 7.4 = 12.2 ± 0.1, being similar to those of the CP1 consensus zinc finger peptide. The specific DNA binding of the protein can be characterized by logβ’pH 7.4 = 8.20 ± 0.08, which is comparable to the affinity of the natural zinc finger proteins (Sp1, WT1, TFIIIA) toward DNA. This value is ~ 1.9 logβ’ unit higher than those determined for semi- or nonspecific DNA binding. Competitive circular dichroism and electrophoretic mobility shift measurements revealed that the conditional stability constant characteristic for Zn(II) binding of 1MEY# protein increased by 3.4 orders of magnitude in the presence of its target DNA sequence.Graphical abstract

Pyrazolones Potentiate Colistin Activity against MCR-1-Producing Resistant Bacteria: Computational and Microbiological Study.

The polymyxin colistin is a last line antibiotic for extensively resistant Gram-negative bacteria. Colistin binding to lipid A disrupts the Gram-negative outer membrane, but mobile colistin resistance (mcr) gene family members confer resistance by catalyzing phosphoethanolamine (PEA) transfer onto lipid A, neutralizing its negative charge to reduce colistin interactions. Multiple mcr isoforms have been identified in clinical and environmental isolates, with mcr-1 being the most widespread and mcr-3 being common in South and East Asia. Preliminary screening revealed that treatment with pyrazolones significantly reduced mcr-1, but not mcr-3, mediated colistin resistance. Molecular dynamics (MD) simulations of the catalytic domains of MCR-1 and a homology model of MCR-3, in different protonation states of active site residues H395/H380 and H478/H463, indicate that the MCR-1 active site has greater water accessibility than MCR-3, but that this is less influenced by changes in protonation. MD-optimized structures of MCR-1 and MCR-3 were used in virtual screening of 20 pyrazolone derivatives. Docking of these into the MCR-1/MCR-3 active sites identifies common residues likely to be involved in protein-ligand interactions, specifically the catalytic threonine (MCR-1 T285, MCR-3 T277) site of PEA addition, as well as differential interactions with adjacent amino acids. Minimal inhibitory concentration assays showed that the pyrazolone with the lowest predicted binding energy (ST3f) restores colistin susceptibility of mcr-1, but not mcr-3, expressing Escherichia coli. Thus, simulations indicate differences in the active site structure between MCR-1 and MCR-3 that may give rise to differences in pyrazolone binding and so relate to differential effects upon producer E. coli. This work identifies pyrazolones as able to restore colistin susceptibility of mcr-1-producing bacteria, laying the foundation for further investigations of their activity as phosphoethanolamine transferase inhibitors as well as of their differential activity toward mcr isoforms.