13C- and 31P-NMR studies of the conformation of carcinogen-modified nucleic acid dimers

The effect of the carcinogen acetylaminofluorene (AAF) on nucleic acid structure was examined using ¹³C- and ³¹P-NMR spectroscopies. Conformational effects were compared in two AAF-modified dinucleoside monophosphates (ApG and GpA) and two AAF-modified deoxydinucleotides (dpApG and dpGpA). Changes in adenine ¹³C chemical shifts on formation of the AAF-adduct and as a function of temperature provided evidence of base stacking. Differences in fluorene ¹³C chemical shifts between the AAF-modified dimer and AAF-modified monomer provided evidence of fluorene stacking. The effect of forming the adduct on the phosphate backbone was examined using ³¹P-NMR. A correlation was demonstrated between the degree of adenine-fluorene stacking on one hand and the change in conformation of the backbone conformation on the other.     

Pregnane X receptor-dependent and -independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation

The arylamide 2-acetylaminofluorene (AAF) is a powerful carcinogen displaying a marked promoting activity, also known to regulate expression of liver detoxifying proteins. In this study we identified CYP3A23, a major inducible cytochrome P-450 (CYP) isoform, as an AAF target in hepatocytes. Indeed, exposure to AAF of primary rat hepatocytes resulted in a marked up-regulation of CYP3A23 expression at both mRNA and protein levels. Using CYP3A23 reporter gene constructs, we further demonstrated that AAF activated the CYP3A23 Direct Repeat 3 (DR3) promoter element interacting with the nuclear pregnane X receptor (PXR). Moreover, the PXR antagonist ecteinascidin-743 fully suppressed AAF-related CYP3A23 induction. Low doses of AAF inhibiting DNA synthesis in hepatocytes however failed to trigger PXR-related CYP3A23 induction and PXR-negative epithelial liver cells remained sensitive to the mito-inhibitory effects of AAF. Such data indicate that AAF up-regulates CYP3A23 through PXR activation but does not require PXR for exerting its carcinogenic promoting properties based on inhibition of cell growth.     

The differential effects of the carcinogen dimethylnitrosamine on isocitrate and 6-phosphogluconate metabolism in single intact cells

A microspectrofluorimetric study is made of the influence of dimethylnitrosamine on NADP reduction, following sequential microinjections into the same L cell, of two substrates: (1) isocitrate, with activity of isocitrate dehydrogenase both in the extramitochondrial and intramitochondrial compartments, (2) 6-phosphogluconate, with activity of the dehydrogenase in the extramitochondrial compartment. In control L cells a two-step reduction of NAD(P) is obtained followed by relatively slow reoxidation. In the minutes which follow addition of carcinogen, e.g., dimethylnitrosamine, to the cell medium the isocitrate and 6-phosphogluconate-induced transient NADP reoxidation is decreased in magnitude compared to control, while the rate constant of NADPH reoxidation is considerably accelerated, possibly due to requirements at the level of the microsomal metabolizing system. Observations within the first hour of carcinogen addition suggest an interesting system for evaluating the immediate actions of carcinogens at extranuclear sites: i.e., a comparative study of NADP reduction-reoxidation rate constants via injection of substrates for extra- vs. intramitochondrial pathways.     

Solid-state 13C-NMR studies of the effects of sodium ions on the gramicidin A ion channel.

End-to-end helical dimers of gramicidin A form transmembrane pores in lipid bilayers, through which monovalent ions may pass. The groups within the peptide that interact with these ions have been studied by application of solid-state spectroscopic methods to a series of gramicidin A analogues synthesized with 13C in selected peptide carbonyl groups. The resonances of D-Leu10, D-Leu12 and D-Leu14 analogues were perturbed in the presence of 0.16 M sodium ions, whereas the resonances of the carbonyls of Gly2, Ala3, D-Leu4 and Val7, which are closer to the formylated N-terminal end of the peptide, were unaffected. The observed changes in chemical shift anisotropy are indicative of a change in orientation of the abovementioned leucine carbonyls.     

Studies on the conformation and dynamics of the C8-substituted guanine adduct of the carcinogen acetylaminofluorene; model for a possible Z-DNA modified structure

The structure of an adduct between guanine and the carcinogen acetylaminofluorene has been examined in the solid state by X-ray crystallography, and in solution by NMR techniques. The observed conformations have been compared with predictions from energy calculations and their relevance to models of adducts with DNA has been examined.     

In vivo 13C-NMR studies on the metabolism of the lugworm Arenicola marina

13C-NMR natural-abundance spectra of specimens of Arenicola marina obtained, showed seasonal changes in the concentration of some metabolites, with the osmolite alanine as well as triacylglyceride storage compounds present at high concentrations. Glycogen was sometimes only barely detectable due to the low natural abundance level of 13C. Glycogenic metabolism of the lugworm A. marina was studied in vivo by 13C-NMR spectroscopy using 13C-labelled glucose. During recovery from a hypoxic period [1-13C]glucose was incorporated into glycogen. [1-13C]Glucose was injected 5 h after the end of hypoxia to guarantee sufficient and reliable 13C labelling of glycogen. An earlier injection of [1-13C]glucose led to considerably diminished incorporation of 13C-labelled glucosyl units into glycogen, probably due to the consumption of the available glucose as fuel for ATP production. No scrambling of 13C into the C6 position of glycogen was observed, indicating a lack of gluconeogenic activity. 13C was also incorporated into the C3 positions of alanine and alanopine. To assign correctly this last 13C-NMR resonance, the compound was synthesized biochemically. No labelling of glycogen was observed when [3-13C]alanine was injected into the coelomic cavity with similar incubation conditions being used. The 13C of [1-13C]glucose, incorporated into glycogen, showed a very low turnover rate in normoxic lugworms as shown by two 13C(1H)-NMR spectra, one obtained 48 h after the other. On the other hand, in hypoxia lugworms the signal due to 13C-labelled glycogen decreased very rapidly proving a high turnover rate. The disappearance of 13C from glycogen during the first 24 h of hypoxia indicates that the last glycosyl units to be synthesized are the first to be utilized. Lugworms were quite sensitive to the 1H-decoupling field used for obtaining the 13C(1H)-NMR spectra, especially at 11.7 T. Using bi-level composite-pulse decoupling and long relaxation delays, no tissue damage or stress-dependent phosphagen mobilization, as judged by 31P-NMR spectroscopy, was observed.     

NMR and CD studies on the interaction of Alzheimer beta-amyloid peptide (12-28) with beta-cyclodextrin

Polymerization of the amyloid beta-peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease (AD). Inhibition of the formation of these toxic polymers of Abeta has emerged as an approach for developing therapeutics for AD. NMR and CD spectra were used to investigate the interaction between cyclodextrin and Abeta(12-28) peptide, which was reported to be an important region for forming amyloid fibrils. CD spectral analyses show that of the alpha-, beta- and gamma-cyclodextrins only beta-cyclodextrin inhibits the aggregation of Abeta(12-28) at pH 5.0. Analysis of the one-dimensional proton NMR spectra of Abeta(12-28) and the mixture of Abeta(12-28) with beta-cyclodextrin clearly indicates that there are chemical shift changes in the aromatic ring of Phe19 and the methyl groups of Val18 in the peptide. The NOESY spectra show cross-peaks between H-3 and H-5 of beta-cyclodextrin and the aromatic protons of Phe19 and Phe20. These chemical shift differences and NOEs demonstrate that there is an interaction between Abeta(12-28) and beta-cyclodextrin. Analysis of the cross-peak intensity in the NOESY spectra reveals that the aromatic rings of Phe19 and 20 are generally inserted into beta-cyclodextrin at the broad side and are oriented toward the narrow side of the cavity.     

Molecular mechanics studies of dinucleoside methylphosphonates: influence of methylphosphonate and its chirality on the phosphodiester conformation.

Molecular mechanics studies are performed on single stranded as well as base paired forms of dinucleoside methylphosphonates comprising different base sequences for both the S- and R-isomers of methylphosphonate (MP). S-MP produces noticeable distortions in the geometry, locally at the phosphate center, and this enables the stereochemical feasibility of compact g- g- phosphodiester. Besides, it tends to perturb the conformations around the P-O3' and glycosyl bonds, causing minor variations in stacking interactions. In single stranded dinucleosides, the gain in adjacent base stacking interaction energies seems to be sufficient to overcome the barrier to P-O3' bond rotation arising due to S-MP...sugar interaction, and this results in transition to a compact phosphodiester (BI-type) from an initial extended phosphodiester (BII-type) conformation. Such a thing seems rather difficult in base pair constrained duplexes. Dinucleosides with R-MP behave analogous to normal phosphate duplexes as the methyl group is away from the sugar. It is found that dinucleoside methylphosphonates are energetically less favoured than the corresponding dinucleoside phosphates mainly due to the depletion of contributions from electrostatic attractive interactions involving the base and sugar with the methylphosphonate consequent to the nonionic nature of the latter. Neither S-MP nor R-MP seem to significantly alter the stereochemistry of duplex structure.     

Direct identification and quantitative determination of costunolide and dehydrocostuslactone in the fixed oil of Laurus novocanariensis by 13C-NMR spectroscopy

The fixed oil of Laurus novocanariensis (previously L. azorica) contains mostly glycerides together with minor non-saponifiable compounds. The direct identification and quantitative determination of costunolide and dehydrocostuslactone, two sesquiterpene lactones components of the oil that exhibit biological activities, is described. The analysis was carried out using 13C-NMR spectroscopy (signal acquisition with inverse gated decoupling of protons; diglyme as internal standard) without separation, derivatisation or any sample preparation.     

Evaluation of steric effects on the exocyclic conformations of 6-C-methyl-substituted 2-acetamido-2-deoxy-beta-D-glucopyranosides

Introduction of a stereodefined methyl group at the C-6 position of N-acetylglucosamine mono- and disaccharides creates a strong and predictable orientational bias on the geminal C-6 hydroxyl in solution, as determined by (1)H-(1)H and (13)C-(1)H NMR coupling constants. The conformational directing effect is more pronounced in the disaccharides because of the greater steric demand imposed by the neighboring glycosidic unit.     

NMR studies on the conformation of oligomannosides and their interaction with banana lectin

The conformational and dynamic behaviour of three mannose containing oligosaccharides, a tetrasaccharide with α1→2, and α1→3, and a penta and a heptasaccharide with α1→2, α1→3, and α1→6 linkages has been evaluated by molecular mechanics and dynamics simulations and NMR spectroscopical methods. It is found that they display a fair amount of conformational freedom, with one major and one minor conformation per glycosidic linkage. The evaluation of their recognition by banana lectin has also been performed by STD NMR methods and a preliminary view of their putative interaction mode has been carried out by means of docking procedures.     

13C-NMR studies on the reaction intermediates of porcine kidney D-amino acid oxidase reconstituted with 13C-enriched flavin adenine dinucleotide

The 13C-NMR spectra of the reaction intermediates of D-amino acid oxidase (DAO) were measured with DAO reconstituted with FAD in which the 2-, 4-, 4a-, and 10a-positions of the isoalloxazine moiety were selectively 13C-enriched. The reaction intermediates used include charge-transfer complexes of the oxidized DAO with substrate intermediates and those of the reduced enzyme with substrate intermediates. For the former type of complex, the reaction intermediates with beta-cyano-D-alanine (D-BCNA) and D-proline were used, while for the latter the purple intermediates with D-alanine and D-proline were chosen. The 13C-resonances of 2-13C in the reaction intermediates with D-BCNA and D-proline were downfield-shifted by about 1 ppm relative to the free oxidized DAO. The 4-13C signal for the DAO-D-BCNA intermediate was observed at 1.2 ppm upfield from that of the oxidized DAO, though that for DAO-D-proline intermediate showed no shift. These results suggest modulation of the hydrogen bondings at C(2) = 0 and/or C(4) = 0 in these reaction intermediates. Comparison of the 13C-resonances of reduced DAO with those of free reduced FMN in the neutral and anionic forms indicate that FAD in reduced DAO is in the anionic reduced form. The 4a-13C resonance of reduced DAO is upfield-shifted by about 3 ppm from that of free reduced anionic FMN. Comparison of the 13C-resonances for the purple intermediates with those of reduced FMN and reduced DAO indicate unequivocally that FAD in the purple intermediate is in the anionic reduced state. The 4a-13C resonances for the purple intermediates were substantially upfield-shifted (by 2.4 ppm with D-alanine and 1.9 ppm with D-proline) relative to reduced DAO. This indicates that the electron density, and hence the nucleophilicity, of the 4a-carbon is elevated in the purple intermediate relative to free reduced DAO. This leads to a model in which the oxidative half reaction proceeds via the reaction of molecular oxygen at the 4a-position of the reduced FAD in the purple intermediate. This provides a rational molecular basis for the oxidative half reaction by way of the purple intermediate prior to product release rather than by way of free reduced enzyme after product release.     

NMR studies of the conformation of the natural sweetener rebaudioside A

Rebaudioside A is a natural sweetener from Stevia rebaudiana in which four β-d-glucopyranose units are attached to the aglycone steviol. Its ¹H and ¹³C NMR spectra in pyridine-d₅ were assigned using 1D and 2D methods. Constrained molecular dynamics of solvated rebaudioside using NMR constraints derived from ROESY cross peaks yielded the orientation of the β-d-glucopyranose units. Hydrogen bonding was examined using the temperature coefficients of the hydroxyl chemical shifts, ROESY and long-range COSY spectra, and proton-proton coupling constants.     

Influence of the glycosidic torsion angle on 13C and 15N shifts in guanosine nucleotides: Investigations of G-tetrad models with alternating syn and anti bases

Summary The effect of the glycosidic torsion angle on ¹³C and ¹⁵N shifts of the sugar and base moieties of guanosine nucleotides was investigated by comparing the sites in two model G-tetrad oligodeoxynucleotides that contain guanosine residues alternately with syn and anti bases. The sugar puckering has been shown to be C2-endo for both cases. It was observed that, for the instances with syn bases, the C1 through C4 carbons showed shifts that may be distinguished from those normally found in B-DNA-like structures. C1, C3 and C4 moved to lower field, while C2 moved to higher field. Effects of the change in glycosidic torsion angle were also seen in the shifts of base carbons and nitrogens in the five-membered ring portion of the base. Characterization of the shift variation associated with this conformational change may be useful in developing the use of ¹³C shifts as a tool in conformational analysis of oligonucleotides.Part of the work reported here derives from the Ph.D. Thesis of Karen L. Greene, Emory University, Atlanta, GA, 1991.     

Thermodynamic studies of binding proteins: effects of temperature variations on substrate binding and conformation of the leucine-isoleucine-valine binding protein of Escherichia coli

The behaviour of the Leucine isoleucine Valine binding protein of Escherichia coli as a function of temperature has been examined. Substrate binding measurements showed a temperature dependence of the leucine-isoleucine-valine binding protein leucine complex formation constants. The protein-substrate complex was completely dissociated beyond 70 degrees C. In the range 5-65 degrees C the protein remained active but Van't Hoff's plots indicated changes of the reaction thermodynamic parameters. Large negative delta Cp values (--2.25 kJ mole-1 K-1 between 5 and 40 degrees C and--9.40 above 40 degrees C) indicate important substrate induced modifications of the protein conformation. Scanning calorimetry of the leucine isoleucine valine binding protein before and after addition of leucine was also performed. Two thermal events were recorded when the protein was substratefree and only one, at a higher temperature and more important, when the substrate was added. The results of these two approaches were in agreement in that both methods suggested a binding dependent conformational change of the protein which resulted in a greater stability of its structure.     

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na⁺/K⁺ ion binding site of the Na,K-ATPase α subunit.     

A molecular mechanics and semiempirical molecular orbital study on the conformation of polynorbornene chains

The conformational analysis of polynorbornene (PNB) chains was investigated with the AM1, MM2, AMBER and OPLS methods taking into consideration the possibility of binding of norbornene monomers to each other at various positions, i.e. exo–exo, exo–endo, endo–endo. The chain that is formed by connecting exo–endo positions of the monomers has lower torsional barrier energy than those formed with bonds at other positions and has more flexibility. It is determined that the thredisyndiotactic chain formed by exo–endo addition adopts a helix structure and has a coil shape. The disyndiotactic chain formed by connecting norbornene monomers in mixed type has a linear structure. It is found that the repeat unit conformations of thredisyndiotactic and disyndiotactic chains of PNB are TGTG^– and (TGTG^–)₂, respectively.     

Triterpenes in the hexane extract of leaves of Olea europaea L.: analysis using 13C-NMR spectroscopy

Two neutral triterpenes and a triterpene acid were identified and quantified directly, in the absence of any purification steps, in a precipitate obtained during the industrial extraction of the leaves of Olea europaea L. using 13C-NMR spectroscopy (spectrometer operating at 4.7 T equipped with a 10 mm probe). The method was optimised in order to reduce the duration of analysis with a routine NMR spectrometer. Together with long-chain linear compounds, erythrodiol, uvaol and oleanolic acid accounted for 27.3, 18.3 and 12.5% of the precipitate, respectively.