Binding Of TNT Research Articles

Docking Troponin T onto the Tropomyosin Overlapping Domain of Thin Filaments

Complete description of thin filament conformational transitions accompanying muscle regulation requires ready access to atomic structures of actin-bound tropomyosin-troponin. To date, several molecular-docking protocols have been employed to identify troponin interactions on actin-tropomyosin because high-resolution experimentally determined structures of filament-associated troponin are not available. However, previously published all-atom models of the thin filament show chain separation and corruption of components during our molecular dynamics simulations of the models, implying artifactual subunit organization, possibly due to incorporation of unorthodox tropomyosin-TnT crystal structures and complex FRET measurements during model construction. For example, the recent Williams et al. (2016) atomistic model of the thin filament displays a paucity of salt bridges and hydrophobic complementarity between the TnT tail (TnT1) and tropomyosin, which is difficult to reconcile with the high, 20 nM Kd binding of TnT onto tropomyosin. Indeed, our molecular dynamics simulations show the TnT1 component in their model partially dissociates from tropomyosin in under 100 ns, whereas actin-tropomyosin and TnT1 models themselves remain intact. We therefore revisited computational work aiming to improve TnT1-thin filament models by employing unbiased docking methodologies, which test billions of trial rotations and translations of TnT1 over three-dimensional grids covering end-to-end bonded tropomyosin alone or tropomyosin on F-actin. We limited conformational searches to the association of well-characterized TnT1 helical domains and either isolated tropomyosin or actin-tropomyosin yet avoided docking TnT domains that lack known or predicted structure. The docking programs PIPER and ClusPro were used, followed by interaction energy optimization and extensive molecular dynamics. TnT1 docked to either side of isolated tropomyosin but uniquely onto one location of actin-bound tropomyosin. The antiparallel interaction with tropomyosin contained abundant salt bridges and intimately integrated hydrophobic networks joining TnT1 and the tropomyosin N-/C-terminal overlapping domain. The TnT1-tropomyosin linkage yields well-defined molecular crevices. Interaction energy measurements strongly favor this TnT1-tropomyosin design over previously proposed models.

Tetraphenylethylene Foldamers with Double Hairpin-Turn Linkers, TNT-Binding Mode and Detection of Highly Diluted TNT Vapor.

Tetraphenylethylene (TPE) foldamers with double hairpin-turn linkers showing an aggregation-induced emission (AIE) effect have been synthesized for the first time. A crystal structure of a foldamer-TNT complex has been obtained, enabling unprecedented direct observation of the interactions between TNT molecules and the chromophores of the foldamer. Instead of π-π stacking interactions, which have often been considered to be the key mechanism in the binding of TNT by chromophoric receptors, strong n-π interactions between the nitro groups of TNT and the aromatic rings of the foldamer have been found. Exceptionally, by addition of 1 % NaF to a suspension of the foldamer in H2 O/THF (95:5), the fluorescence quenching efficiency by TNT vapor significantly increased from about 20 % to more than 90 %. Even after diluting TNT-saturated air at 25 °C by a factor of 2×104 , an obvious quenching response was observed, indicating that ultratrace TNT vapor (down to 3.4 fg per mL of air) could be detected.

Sensitive Detection of TNT using Competition Assay on Quartz Crystal Microbalance

Currently available TNT sensors are characterized by high sensitivity, but low specificity, which limits the detection of TNT in dirty environments. We report here a TNT sensor designed to measure the displacement of a TNT-specific antibody by quartz crystal microbalance (QCM). This sensor combines high sensitivity of detection (0.1 ng/mL) with the ability to distinguish TNT from molecules with similar chemical properties. Particularly, the reliability of this method for the detection of TNT in dirty environments was investigated by using fertilizer solution and artificial seawater. Instead of measuring actual binding of TNT, the method described is based on the displacement of an anti-TNT antibody, which allows quantifying the concentration of TNT in solution with higher sensitivity. In addition, by utilizing the rate of antibody displacement, the detection time is significantly decreased from hours, which would be necessary to measure the frequency change at equilibrium, to minutes. A Langmuir kinetic model was used to describe the molecular interactions on the surface of the sensor and to establish a standard curve to estimate on-site TNT detection. In summary, QCM detection of anti-TNT antibody displacement provides a method for rapid detection of TNT with high sensitivity and specificity.

Binding of TNT to amplifying fluorescent polymers: An ab initio and molecular dynamics study

Molecular modeling techniques were employed to study the interaction of trinitrotoluene with an amplifying fluorescent polymer used in explosive sensor devices. The pentiptycene moiety present in these polymers appears to be the most energetically favorable binding site for trinitrotoluene. Surface features of the polymer suggest that the small cavity feature of the pentiptycene moiety may be more available for binding to analyte compounds due to steric crowding about the large cavity. Binding energies between model binding sites of the polymer and various analyte compounds were more rigorously estimated by semiempirical and ab initio techniques. Binding energies were found to be largest with trinitrotoluene and other nitroaromatic compounds. Electrostatic and π-stacking interactions between trinitrotoluene and the model host were investigated by studying a series of modified host compounds.

Ultrasensitive Surface Plasmon Resonance Detection of Trinitrotoluene by a Bis-aniline-Cross-Linked Au Nanoparticles Composite

A bis-aniline-cross-linked Au nanoparticles (NPs) composite is electropolymerized on Au surfaces. The association of trinitrotoluene, TNT, to the bis-aniline bridging units via pi-donor-acceptor interactions allows the amplified detection of TNT by following the surface plasmon resonance (SPR) reflectance changes as a result of the coupling between the localized plasmon of the AuNPs and the surface plasmon wave associated with the gold surface. The detection limit for analyzing TNT by this method is approximately 10 pM. The electropolymerization of the bis-aniline-cross-linked AuNPs composite in the presence of picric acid results in a molecular-imprinted matrix for the enhanced binding of TNT. The imprinted AuNPs composite enabled the sensing of TNT with a detection limit that corresponded to 10 fM. Analysis of the SPR reflectance changes in the presence of different concentrations of TNT revealed a two-step calibration curve that included the ultrasensitive detection of TNT by the imprinted sites in the composite, KassI. for the association of TNT to the imprinted sites, 6.4 x 10(12) M-1, followed by a less sensitive detection of TNT by the nonimprinted pi-donor bis-aniline sites (KNIass. = 3.9 x 10(9) M-1). The imprinted AuNPs composite reveals impressive selectivity. The structural and functional features of the bis-aniline-cross-linked AuNPs composites were characterized by different methods including ellipsometry, AFM, and electrochemical means. The dielectric properties of the AuNPs composite in the presence of different concentrations of TNT were evaluated by the theoretical fitting of the respective experimental SPR curves. The ultrasensitive detection of the TNT by the AuNPs composite was attributed to the changes of the dielectric properties of the composite, as a result of the formation of the pi-donor-acceptor complexes between TNT and the bis-aniline units. These changes in the dielectric properties lead to a change in the conductivity of the AuNPs matrix.

Pattern recognition based identification of nitrated explosives

We report an effective approach for the identification of nitro explosives by the reaction of an electron-acceptor with an electron-donor with and without the addition of γ-cyclodextrin (γ-CD). A charge-transfer complex was prepared in acetonitrile by the reaction of the nitro explosive TNT as an electron acceptor with the donor N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). The complex TNT/TMPD converts gradually into TNT˙−/TMPD˙+ in water with 5% acetonitrile by volume. The inclusion behavior of TNT˙−/TMPD˙+ with γ-CD was studied by UV-visible spectroscopy. The effect of α- and β-CD with TNT˙−/TMPD˙+ was also studied. Similar experiments were also carried out for RDX˙−/TMPD˙+ with γ-CD. The binding of TNT˙−/TMPD˙+ and RDX˙−/TMPD˙+ with γ-CD were compared for the application of the complexes in a sensor array. Subsequently, the visible absorption spectra of nitro explosives as analytes, such as TNT, RDX, HMX and Tetryl with TMPD in the presence and absence of γ-CD, were measured with time. We worked with the same analytes with a second electron donor tetrakis(dimethylamino)ethylene (TDAE) using 96-well plate experiments. Pattern recognition analysis was applied to analyze the data collected and it showed successful discrimination of the nitro explosives. This work represents the simplest colorimetric chemical sensor technique to be used in monitoring the range of common nitro explosives.

Early events in 2,4,6-trinitrotoluene (TNT) degradation by porphyrins: Binding of TNT to porphyrin by hydrophobic and hydrogen bonds

The interaction of meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl) porphyrin (C 1TPP) with 2,4,6-trinitrotoluene (TNT) has been explored by UV–vis and fluorescence spectroscopy. The influence of temperature on the interaction has also been studied. C 1TPP binds to TNT at pH 7.0 at room temperature via 1.94 kcal/mole hydrogen bonds with absorbance loss at 412–413 nm and the appearance of a new peak at 422–424 nm. The hydrogen binding of TNT to C 1TPP was confirmed by the dissolution of the complex upon the addition of urea. Increasing the temperature results in the appearance of a new absorbance peak at 540 nm and absorbance loss at 515 nm with activation energy of 29.7 kcal/mole in the range of the hydrophobic bond energy. This suggests the hydrophobic bonding of TNT with the pyrrole nitrogens in the porphyrin. Increasing the concentration of the TNT in the solution quenches the fluorescence of the porphyrin following the Stern–Volmer equation. The association constants calculated from absorbance and fluorescence are expectedly similar.

Sunlight-catalyzed conversion of cyclic organics with novel mesoporous organosilicas

Abstract Porphyrin-embedded periodic mesoporous organosilica (PMO) materials were developed for sunlight-stimulated degradation of cyclic organics. The binding affinity and selectivity of the PMOs act to bring porphyrin and target into close proximity, provide extensive surface area, and a stable substrate for porphyrin immobilization. Aqueous samples incubated with these PMO materials showed a decrease in analyte concentration greater than that expected based on adsorption and uncatalyzed photoconversion during 5 h of sunlight illumination. Both materials show selective binding of TNT over structurally similar compounds such as p -nitrophenol. Template directed molecular imprinting was used in an attempt to increase the selectivity of the catalyst.

Binding of 2,4,6-trinitrotoluene, aniline, and nitrobenzene to dissolved and particulate soil organic matter.

The distribution of TNT* (the sum of TNT and its degradation products), aniline, and nitrobenzene between particulate organic matter (POM), dissolved soil organic matter (DOM), and free compound was studied in controlled kinetic (with and without irradiation) and equilibrium experiments with mixtures of POM and DOM reflecting natural situations in organic rich soils. The binding of TNT* to POM was fast, independent of irradiation, and adsorption isotherms had a great linear contribution (as determined by a mixed model), indicative of a hydrophobic partitioning mechanism. The binding of TNT* to DOM was slower, strongly enhanced under nonirradiated conditions, and adsorption isotherms were highly nonlinear, indicative of a specific interaction between TNT derivatives and functional groups of DOM. Nitrobenzene was associated to both POM and DOM via hydrophobic partitioning, whereas aniline binding was dominated by specific binding to POM and DOM functional groups. On the basis of nitrobenzene and TNT* adsorption parameters determined by a mixed Langmuir + linear model, POM had 2-3 times greater density of hydrophobic moieties as compared to DOM. This difference was reflected by a greater (O + N)/C atomic ratio for DOM. The sum of C-C and C-H moieties, as determined by X-ray photoelectron spectroscopy (XPS), and the sum of aryl-C and alkyl-C, as determined by solid-state cross-polarization magic-angle spinning (CP-MAS) 13C NMR, could only qualitatively account for differences in adsorption parameters. Aliphatic C was found to be more important for the hydrophobic partitioning than aromatic C. On the basis of nonlinear adsorption parameters,the density of functional groups reactive with aniline and TNT derivatives was 1.3-1.4 times greater in DOM than in POM, which was in fair agreement with 13C NMR and XPS data for the sum of carboxyl and carbonyl groups as potential sites for electrostatic and covalent bonding. We conclude that in contaminated soils characterized by continuous leaching of DOM, formation of TNT derivatives (via biotic and abiotic reductive degradation) and their preference for specific functional groups in DOM may contribute to a significant transportation of potentially toxic TNT compounds into surface waters and groundwaters.

15N NMR investigation of the reduction and binding of TNT in an aerobic bench scale reactor simulating windrow composting.

T15NT was added to a soil of low organic carbon content and composted for 20 days in an aerobic bench scale reactor. The finished whole compost and fulvic acid, humic acid, humin, and lignocellulose fractions extracted from the compost were analyzed by solid-state CP/MAS and DP/MAS 15N NMR. 15N NMR spectra provided direct spectroscopic evidence for reduction of TNT followed by covalent binding of the reduced metabolites to organic matter of the composted soil, with the majority of metabolite found in the lignocellulose fraction, by mass also the major fraction of the compost. In general, the types of bonds formed between soil organic matter and reduced TNT amines in controlled laboratory reactions were observed in the spectra of the whole compost and fractions, confirming that during composting TNT is reduced to amines that form covalent bonds with organic matter through aminohydroquinone, aminoquinone, heterocyclic, and imine linkages, among others. Concentrations of imine nitrogens in the compost spectra suggest that covalent binding by the diamines 2,4DANT and 2,6DANT is a significant process in the transformation of TNT into bound residues. Liquid-phase 15N NMR spectra of the fulvic acid and humin fractions provided possible evidence for involvement of phenoloxidase enzymes in covalent bond formation.

Mapping the Interacting Regions between Troponins T and C: BINDING OF TnT AND TnI PEPTIDES TO TnC AND NMR MAPPING OF THE TnT-BINDING SITE ON TnC

Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex. The interactions among the components of the troponin complex (troponins T, C, and I) are essential to understanding the regulation of muscle contraction. While the structure of TnC is well known, and a model for the binary TnC.TnI complex has been recently published (Tung, C.-S., Wall, M. E., Gallagher, S. C., and Trewhella, J. (2000) Protein Sci. 9, 1312-1326), very little is known about TnT. Using non-denaturing gels and NMR spectroscopy, we have analyzed the interactions between TnC and five peptides from TnT as well as how three TnI peptides affect these interactions. Rabbit fast skeletal muscle peptide TnT-(160-193) binds to TnC with a dissociation constant of 30 +/- 6 microm. This binding still occurs in the presence of TnI-(1-40) but is prevented by the presence of TnI-(56-115) or TnI-(96-139), both containing the primary inhibitory region of TnI. TnT-(228-260) also binds TnC. The binding site for TnT-(160-193) is located on the C-terminal domain of TnC and was mapped to the surface of TnC using NMR chemical shift mapping techniques. In the context of the model for the TnC.TnI complex, we discuss the interactions between TnT and the other troponin subunits.

Solid-state 2H MAS NMR studies of TNT absorption in soil and clays.

The binding of TNT to a soil and soil components is studied with 2H MAS NMR. This NMR technique clearly shows different interactions for deuterated TNT with soil, quartz, and clays. Basically, this NMR method relies on the well-defined 2H MAS NMR characteristics of a deuterated methyl group attached to an aromatic ring. Stationary rings and rings executing 2- or 3-fold jump motions all yield different NMR spectra. The technique has good sensitivity; these studies were conducted at a TNT loading of 22 mmol/kg. For the soil sample studied, TNT showed binding similar to a TNT/K10-montmorillonite sample.

Mechanism of bioactivation and covalent binding of 2,4,6-trinitrotoluene

Studies were undertaken to investigate the mechanism of bioactivation and covalent binding of TNT. Incubation of [ 14C]TNT with rat liver microsomes in the presence of an NADPH generating system resulted in metabolism and covalent binding to microsomal proteins. Timedependence studies showed that TNT was rapidly reduced to yield 4-hydroxylamino-2,6-dinitrotoluene (4HA), 4-amino-2,6-dinitrotoluene (4A) and 2-amino-4,6-dinitrotoluene (2A) as intermediates which were further metabolized to form 2,4-diamino-6-nitrotoluene (2,4DA) and 2,6-diamino-4-nitrotoluene (2,6DA). In contrast to the rapid disappearance of TNT, formation of covalent protein adducts increased with time, suggesting that the reactive intermediate was likely to be formed not directly from TNT but from proximal intermediates such as 4HA. The hypothesis that 4HA was more readily converted to the reactive intermediate than TNT was further supported by the increased levels of covalent adduct formation when [ 14C]4HA was incubated directly with liver microsomes. Covalent binding of TNT and 4HA was dependent on oxygen concentration. Higher levels of covalent adducts were formed when TNT was incubated aerobically (up to 50% oxygen concentration) than under anaerobic conditions. Covalent binding of [ 14C]4HA also increased with increasing oxygen concentrations. These results suggest that the reactive intermediate is likely to be an oxidized metabolite of 4HA, e.g. 4-nitroso-2,6-dinitrotoluene. Compounds containing a free sulfhydryl group (cysteine, N-acetylcysteine, GSH or 3,4-dichlorobenzenethiol) decreased the amount of covalent binding to various degrees, suggesting the involvement of the sulfhydryl group in adduct formation with TNT following bioactivation. Metabolic activation of TNT by liver microsomes required NADPH but not NADH as the cofactor. Incubation of [ 14C]TNT with purified rat liver NADPH cytochrome P450 reductase under either aerobic or anaerobic conditions yielded exclusively 4HA. In contrast, 2A and 4A were formed following incubation of TNT with the reconstituted system containing cytochrome P450, NADPH cytochrome P450 reductase and dilauroyl phosphatidylcholine. These observations suggest that the initial reduction of the nitro group can be catalyzed by NADPH cytochrome P450 reductase alone but cytochrome P450 is needed in the reduction of the hydroxylamine to the amine.