Stable Isotope Labeling Strategy for Protein–Ligand Binding Analysis in Multi-Component Protein Mixtures

Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein–ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein–ligand binding interactions. The protocol utilizes an H₂¹⁶O₂ and H₂¹⁸O₂ labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein–ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the ¹⁸O/¹⁶O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique’s ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein–protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).     

Experimental and Computational Models for Side Chain Discrimination in Peptide–Protein Interactions

A bis(18-crown-6) Tröger's base receptor and 4-substituted hepta-1,7-diyl bisammonium salt ligands have been used as a model system to study the interactions between non-polar side chains of peptides and an aromatic cavity of a protein. NMR titrations and NOESY/ROESY NMR spectroscopy were used to analyze the discrimination of the ligands by the receptor based on the substituent of the ligand, both quantitatively (free binding energies) and qualitatively (conformations). The analysis showed that an all-anti conformation of the heptane chain was preferred for most of the ligands, both free and when bound to the receptor, and that for all of the receptor-ligand complexes, the substituent was located inside or partly inside of the aromatic cavity of the receptor. We estimated the free binding energy of a methyl- and a phenyl group to an aromatic cavity, via CH-π, and combined aromatic CH-π and π-π interactions to be −1.7 and −3.3 kJ mol⁻¹, respectively. The experimental results were used to assess the accuracy of different computational methods, including molecular mechanics (MM) and density functional theory (DFT) methods, showing that MM was superior.     

PepPro: A Nonredundant Structure Data Set for Benchmarking Peptide–Protein Computational Docking

We present a nonredundant benchmark, coined PepPro, for testing peptide–protein docking algorithms. Currently, PepPro contains 89 nonredundant experimentally determined peptide–protein complex structures, with peptide sequence lengths ranging from 5 to 30 amino acids. The benchmark covers peptides with distinct secondary structures, including helix, partial helix, a mixture of helix and β-sheet, β-sheet formed through binding, β-sheet formed through self-folding, and coil. In addition, unbound proteins' structures are provided for 58 complexes and can be used for testing the ability of a docking algorithm handling the conformational changes of proteins during the binding process. PepPro should benefit the docking community for the development and improvement of peptide docking algorithms. The benchmark is available at http://zoulab.dalton.missouri.edu/PepPro_benchmark . © 2019 Wiley Periodicals, Inc.     

Mixed Metal–Organic Framework with Multiple Binding Sites for Efficient C2H2/CO2 Separation

The separation of C₂H₂/CO₂ is particularly challenging owing to their similarities in physical properties and molecular sizes. Reported here is a mixed metal–organic framework (M′MOF), [Fe(pyz)Ni(CN)₄] ( FeNi-M′MOF , pyz=pyrazine), with multiple functional sites and compact one-dimensional channels of about 4.0 Å for C₂H₂/CO₂ separation. This MOF shows not only a remarkable volumetric C₂H₂ uptake of 133 cm³ cm⁻³, but also an excellent C₂H₂/CO₂ selectivity of 24 under ambient conditions, resulting in the second highest C₂H₂-capture amount of 4.54 mol L⁻¹, thus outperforming most previous benchmark materials. The separation performance of this material is driven by π–π stacking and multiple intermolecular interactions between C₂H₂ molecules and the binding sites of FeNi-M′MOF . This material can be facilely synthesized at room temperature and is water stable, highlighting FeNi-M′MOF as a promising material for C₂H₂/CO₂ separation.     

The CH–π Interaction in Protein–Carbohydrate Binding: Bioinformatics and In Vitro Quantification

The molecular recognition of carbohydrates by proteins plays a key role in many biological processes including immune response, pathogen entry into a cell, and cell–cell adhesion (e.g., in cancer metastasis). Carbohydrates interact with proteins mainly through hydrogen bonding, metal-ion-mediated interaction, and non-polar dispersion interactions. The role of dispersion-driven CH–π interactions (stacking) in protein–carbohydrate recognition has been underestimated for a long time considering the polar interactions to be the main forces for saccharide interactions. However, over the last few years it turns out that non-polar interactions are equally important. In this study, we analyzed the CH–π interactions employing bioinformatics (data mining, structural analysis), several experimental (isothermal titration calorimetry (ITC), X-ray crystallography), and computational techniques. The Protein Data Bank (PDB) has been used as a source of structural data. The PDB contains over 12 000 protein complexes with carbohydrates. Stacking interactions are very frequently present in such complexes (about 39 % of identified structures). The calculations and the ITC measurement results suggest that the CH–π stacking contribution to the overall binding energy ranges from 4 up to 8 kcal mol⁻¹. All the results show that the stacking CH–π interactions in protein–carbohydrate complexes can be considered to be a driving force of the binding in such complexes.     

Halogen–C2H2 Binding in Ultramicroporous Metal–Organic Frameworks (MOFs) for Benchmark C2H2/CO2 Separation Selectivity

Acetylene (C₂H₂) capture is a step in a number of industrial processes, but it comes with a high-energy footprint. Although physisorbents have the potential to reduce this energy footprint, they are handicapped by generally poor selectivity versus other relevant gases, such as CO₂ and C₂H₄. In the case of CO₂, the respective physicochemical properties are so similar that traditional physisorbents, such as zeolites, silica, and activated carbons cannot differentiate well between CO₂ and C₂H₂. Herein, we report that a family of three isostructural, ultramicroporous (<7 Å) diamondoid metal–organic frameworks, [Cu(TMBP)X] (TMBP=3,3′,5,5′-tetramethyl-4,4′-bipyrazole), TCuX (X=Cl, Br, I), offer new benchmark C₂H₂/CO₂ separation selectivity at ambient temperature and pressure. We attribute this performance to a new type of strong binding site for C₂H₂. Specifically, halogen ⋅⋅⋅ HC interactions coupled with other noncovalent in a tight binding site is C₂H₂ specific versus CO₂. The binding site is distinct from those found in previous benchmark sorbents, which are based on open metal sites or electrostatic interactions enabled by inorganic fluoro or oxo anions.     

PMA-SiO2–Mediated MCR in PEG-400: A Greener aza-Friedel–Crafts Reaction Leading to 3-Arylmethyl/Diarylmethyl Indoles

A greener approach for the synthesis of 3-arylmethyl/diarylmethyl indoles has been achieved via a PMA-SiO₂-mediated three-component reaction (the aza-Friedel–Crafts reaction) involving indoles, aldehydes, and N,N-disubstituted anilines in PEG-400. A variety of indole derivatives were prepared by using this operationally simple and straightforward methodology in acceptable yields.     

Simultaneous Quantification of Benazepril, Gliclazide and Valsartan in Human Plasma by LC–MS–MS and Application for Rapidly Measuring Protein Binding Interaction between Rhein and These Three Drugs

For new drug candidates with high protein binding in the treatment of diabetic nephropathy, their influence on the protein bindings of angiotensin-converting enzyme inhibitors, sulfonylurea drugs, and angiotensin receptor blockers should be predicted to prevent side effects. To provide an efficient tool for this study, a sensitive and rapid LC–MS–MS method was developed for the simultaneous quantification of representative drugs, benazepril, gliclazide and valsartan in human plasma. Chromatographic separation was performed on a Shim-pack VP-ODS C18 column (250 × 2.0 mm i.d., 5 μm) using methanol–0.05% formic acid (90:10, v/v) as mobile phase. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in SRM mode. Lower limits of quantification were 2, 2 and 20 ng^?1 mL for benazepril, gliclazide and valsartan with 0.1 mL plasma sample. The method fulfills the precision, accuracy, linearity, sensitivity, selectivity requirements to quantify the three drugs and has been successfully used to studying protein binding of benazepril, gliclazide and valsartan in the presence of rhein.     

Galectin–Glycan Interactions: Guidelines for Monitoring by 77Se NMR Spectroscopy,and Solvent (H2O/D2O) Impact on Binding

Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance. Their study is facilitated by nonhydrolysable derivatives of the natural O-glycans, such as S- and Se-glycosides. The latter enable extensive analyses by specific ⁷⁷Se NMR spectroscopy, but still remain underexplored. By using the example of selenodigalactoside (SeDG) and the human galectin-1 and -3, we have evaluated diverse ⁷⁷Se NMR detection methods and propose selective ¹H,⁷⁷Se heteronuclear Hartmann–Hahn transfer for efficient use in competitive NMR screening against a selenoglycoside spy ligand. By fluorescence anisotropy, circular dichroism, and isothermal titration calorimetry (ITC), we show that the affinity and thermodynamics of SeDG binding by galectins are similar to thiodigalactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact. ITC data in D₂O versus H₂O are similar for TDG and LacNAc binding by both galectins, but a solvent effect, indicating solvent rearrangement at the binding site, is hinted at for SeDG and clearly observed for LacNAc dimers with extended chain length.     

A theoretical evidence for cooperativity effects in fluorine-centered halogen bonds: linear (FCN)2–7 and (FNC)2–7 clusters

A theoretical investigation was performed to study cooperative effects in fluorine-centered halogen bond interactions. We investigated geometry, strength, and origin of the interactions in linear (FCN)_2–7 and (FNC)_2–7 clusters by means of MP2 and CCSD(T) methods. Our results strongly suggest that cooperative effects induced by fluorine-centered halogen bonds are significant in both linear FCN and FNC clusters. CCSD(T)/6-311++G** calculations reveal that for (FCN)_2–7 clusters, the average halogen-bonding energy per monomer increases from ?0.76 kcal/mol in dimer to ?0.92 kcal/mol in heptamer. The results of electron density analysis suggest that the capacity of the linear FCN and FNC clusters to concentrate electrons at the F···N and F···C BCPs enhance considerably with cluster size. The results also indicate that the magnitude of cooperative effects is more important for FCN than for FNC clusters. According to energy decomposition analysis, attractive electrostatic and dispersion components make the major contribution to the F···N and F···C halogen bond interactions. An acceptable correlation is found between different energy terms and total interaction energies, revealing the main role of these interactions for stability of linear (FCN) _n and (FNC) _n clusters.     

Solid-Phase Surface Adsorption in Ag–Au|F–, H2O and Ag–Au|ClO–4, H2O Systems: Allowing for the Solid-Solution Nonideality

Integral and differential (with respect to the composition) isotherms of changes in the interfacial free energy (_m– ), the charge density q, and the surface composition X _Au of alloys equilibrated with an aqueous surface-inactive electrolyte are obtained in terms of a finite-thickness interfacial layer, with use of concentration dependences of activity coefficients of components of a polycrystalline binary alloy. Using ac measurements of the double-layer parameters, it is stated that the surface-active component in the Ag–Au|F^–, H₂O and Ag–Au|ClO^– ₄, H₂O systems at 298 K is gold. The Ag–Au solid solution shows negative deviations from Raoult's law, except for the compositions X _Au 0.04 and X _Au 0.80, where the solid solution properties approach those of an ideal solution.     

Minoxidil–2-hydroxypropyl-β-cyclodextrin inclusion complexes: characterization and in vivo evaluation of an aqueous solution for hair growth in rats

Minoxidil dissolved in organic solvents is commonly used in the treatment of androgenic alopecia because it is hydrophobic and poorly soluble in water. The aim of this investigation was to develop an aqueous minoxidil solution without addition of organic solvents. To this end, minoxidil was encapsulated in 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) to form a minoxidil–HP-β-CD inclusion complex with aqueous solubility higher than that of pure minoxidil. Minoxidil–HP-β-CD inclusion complexes were prepared using a freeze-drying method from minoxidil and HP-β-CD at a molar ratio of 1:1. Complex formation was confirmed by nuclear magnetic resonance, thermogravimetric analysis, and thin layer chromatography. The structure of the complex was determined using two-dimensional rotating frame overhauser effect spectroscopy. Minoxidil–HP-β-CD inclusion complexes were dissolved in water to form a homogeneous aqueous solution, and its effect on the hair cycle of mice was evaluated in vivo. The results support the feasibility of using HP-β-CD to prepare an aqueous minoxidil solution to replace organic solvent-based solutions.     

Uranium substitution for tungsten in the Bi2WO6–Bi2UO6 system: Formation of a broad high-temperature solid solution

Polythermic studies of Bi₂WO₆–Bi₂UO₆ system revealed existence of a broad Bi₂W_1?xU_xO₆ solid solution with the Aurivillius-type structure. Below 750 °C, the homogeneity area is narrow (x ≤ 0.12) but extends significantly at higher temperatures (up to ca. 75% Bi₂UO₆ at 1025 °C). The solid solution retains orthorhombic symmetry over the entire compositional range, but substitution of U for W results in significant lowering of second harmonic intensity and Curie point suggesting, at high substitution degrees, a very close approach to the archetypic body-centered tetragonal centrosymmetric structure. The known Bi₂WO₆-based solid solutions are discussed in terms of thermal stability, and a general geometrical explanation is suggested. Incorporation of large cations into the B perovskitic positions of [Bi₂O₂][A_n?1B_nO_3n+1] Aurivillius structures seems yet to be restricted by the n = 1 case.     

The importance of 1:1 and 1:2 metal–ligand species in chiral copper(II)–bis(oxazoline) complexes for catalytic activity

The rate ratio of different copper(II)–bis(oxazoline) complexes for catalytic activity was assessed in a simple competitive experiment. The cumulated product enantioselectivity derived from a mixture of catalysts with antipodal chiral induction provided sufficient information to determine the relative rates in the asymmetric Friedel–Crafts alkylations of indoles with benzylidene malonates. The discovery of non-linear effects in the reaction system investigated gave insights into the active and resting states of the catalysts. This approach can serve as a tool to screen the activities of different catalysts when detailed kinetic measurements are not suggestive.     

Deprotection Chemistry Mediated by ZrOCl2 · 8H2O: An Efficient,Mild, and Green Method for the Conversion of Oximes to Carbonyl Compounds in Aqueous Acetone

Abstract

Less-toxic, moisture-stable, inexpensive, and ecofriendly zirconium oxychloride octahydrate (ZrOCl₂ · 8H₂O) in aqueous acetone (1:1) mediates the conversion of oximes to carbonyl compounds in moderate to good yields. This green methodology is applicable to both aldoximes and ketoximes with tolerance to >C?C<, -NO₂, -OH, and -Cl groups. The reaction and workup are simple.

Fe2O3@[proline]–CuMgAl–LDH: A magnetic bifunctional copper and organocatalyst system for one-pot synthesis of quinolines and 2H-indazoles in green media

A novel magnetic core–shell Fe₂O₃@[proline]–CuMgAl–L(ayered)D(ouble)H(ydroxide) was designed as an efficient bifunctional catalytic system. To this end, Cu (II) was combined with Mg and Al in the LDH structure and l -proline was intercalated between LDH layers in order to perform a straightforward synthesis of quinolines and 2H-indazoles as two important pharmaceutical N-aryl-substituted heterocyclic compounds. In this regard, a facile method was employed through consecutive condensation under a mild conditions in choline azide media, which played the role of a reagent and a solvent to avoid toxic solvents and hazardous azidation reagents. These techniques provided considerable improvement in terms of using green media, reducing starting materials, reaching higher yields and offering a shorter reaction time and lower temperature. In conclusion, it was found that the catalyst could be reused five times with no significant loss of activity.     

Thermodynamic Model for Amorphous Pd(OH)2 Solubility in the Aqueous Na+–K+–H+–OH?–Cl?–ClO 4 ? –H2O System at 25?°C: A Critical Review

Only a limited number of experimental investigations have been conducted to determine hydrolysis constants of Pd(II) or the solubility product of Pd(II) hydroxide, and the reported values differ considerably from each other. No comprehensive reliable thermodynamic model is available for the Pd?COH and Pd?CCl systems. To obtain such a model, thermodynamic data for palladium compounds and complexes with chloride and hydroxide were critically evaluated using the SIT model. These evaluations, in most cases, involved reinterpretation of the original data reported in various publications to produce values of equilibrium constants for various reactions that are consistent with all of available reliable literature data. Final recommended values for solubility products and complexes of Pd(II) with hydroxide and chloride, along with the values for SIT ion interaction parameters, are tabulated.     

A model for predicting the solubility of 1,3,5-trinitro-1,3,5-s-triazine (RDX) in supercritical CO2: isothermal–isobaric Monte Carlo simulations

Isothermal–isobaric Monte Carlo (NPT-MC) simulations and the Widom test particle method were used to predict the solubility of the explosive 1,3,5-trinitro-1,3,5-s-triazine (RDX) in supercritical CO₂. A Lennard–Jones potential energy function was chosen to describe the interaction between RDX and CO₂, and calibrated using two experimental solubility values. NPT-MC simulations using this interaction potential predicted solubilities of RDX in CO₂ over a temperature range of 308 to 353 K and pressures ranging from 10.4 to 48.3 MPa that were in good agreement with experimental values.     

Cs–Ba separation using N2O as a reactant gas in a Multiple Collector-Inductively Coupled Plasma Mass Spectrometer collision-reaction cell: Application to the measurements of Cs isotopes in spent nuclear fuel samples

In the general frameworks of the nuclear fuel cycle and environmental research field, the Cs isotopic composition must be known with high precision and accuracy. The direct determination of Cs isotopes by mass spectrometry techniques is generally hampered by the presence of Ba isobaric interferences however. Here we present a new method which takes advantage of the collision-reaction cell based Multiple Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICPMS) and allows to analyse Cs isotopes in the presence of Ba without prior separation step. The addition of N₂O gas in the cell leads to an antagonistic behavior of Cs⁺ and Ba⁺ as the latter reacts with the gas to form BaO⁺ and BaOH⁺ products whereas Cs⁺ remains unreactive. The efficiency of the method was demonstrated for an UOx sample by comparing the results obtained (1) from the measurements of pure Cs fractions and (2) from Fission Products fractions containing more than 30 ionisable elements in addition to Cs, Ba, and where U and Pu were previously removed by using ion exchange resin. An excellent agreement is achieved between each set of experiments with an external reproducibility always better than 0.5% (RSD, k = 2). This study confirms the strong potential of collision–reaction cell to measure Cs isotopes in presence of interfering Ba, precluding therefore former systematic chemical separations.     

Innovative direct introduction-ion mobility–mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix: Application to 2′-fucosyllactose (2’-FL) in breast milk

Identification and specific quantification of isomers in a complex biological matrix by mass spectrometry alone is not an easy task due to their identical chemical formula and therefore their same mass-to-charge ratio (m/z). Here, the potential of direct introduction combined with ion mobility–mass spectrometry (DI-IM-MS) for rapid quantification of isomers as human milk oligosaccharides (HMOs) was investigated. Differences in HMO profiles between various analyzed breast milk samples were highlighted using the single ion mobility monitoring (SIM²) acquisition for high ion mobility resolution detection. Furthermore, the Se+ (secretor) or Se− (non-secretor) phenotype could be assigned to breast milk samples studied based on their HMO contents, especially on the response of 2′-fucosyllactose (2’-FL) and lacto-N-fucopentaose I (LNFP I). The possibility of quantifying a specific isomer in breast milk by DI-IM-MS was also investigated. The standard addition method allowed the determination of the 2’-FL despite the presence of other oligosaccharides, including 3-fucosyllactose (3-FL) isomer in breast milk. This proof-of-concept study demonstrated the high potential of such an approach for the rapid and convenient quantification of isomers in complex mixtures.