The Hunt for the Closed Conformation of the Fruit-Ripening Enzyme 1-Aminocyclopropane-1-carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study

1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.     

Recent advances in amino acid analysis by capillary electrophoresis

This paper describes a number of articles that have been published on amino acid analysis using CE during the period from June 2003 to May 2005. This review article follows the previous ones of Smith (Electrophoresis 1999, 20, 3078-3083), Prata et al. (Electrophoresis 2001, 22, 4129-4138), and Poinsot et al. (Electrophoresis 2003, 24, 4047-4062). Several new developments in amino acid analysis with CE are reported concerning UV detection, LIF, MS, and NMR. In addition, we describe articles concerning clinical and pharmaceutical studies, neuroclinical applications, and agricultural and food analysis.     

pH dependence of the interaction between rhodamine B and the water-soluble poly(sodium 4-styrenesulfonate)

The binding of rhodamine B (RB) to the polyanion containing aromatic groups poly(sodium 4-styrenesulfonate) (PSS) is studied by separation and spectroscopic techniques at pH between 2 and 7. Significant binding is found at pH below 5, together with a red-shift of the RB maximum of absorbance to 564 nm, and RB fluorescence quenching. The dependence of the pH is related with protonation of RB molecules. Fluorescence quenching is a consequence of a more hydrophobic environment and may occur on territorially or site-specifically bound molecules, and/or on self-aggregated molecules in a hydrophobic polymer domain. Remarkably, the basicity of RB is increased by the influence of the polymer.     

Enrichment of the glycoalkaloids alpha-solanine and alpha-chaconine from potato juice by adsorptive bubble separation using a pH gradient

For the first time, the solanidine alkaloids alpha-solanine and alpha-chaconine could be quantitatively enriched from potato juice by Adsorptive Bubble Separation (ABS) with a pH gradient. The enrichment into the foam was influenced by the pH value, bubble size, and gas flow rate. The efficiency was highest on using diluted samples with a concentration between 2 and 6 mg L(-1) of the alkaloids at pH 6.0. The experiments with a standard solution of each alkaloid confirmed that these substances can be quantitatively enriched into the 'spumat' without surface active potato proteins. The transfer into the foam fraction under these conditions was similar to that from the aqueous potato extract.     

Intraspecific variability of carbon isotope discrimination and its correlation with grain yield in safflower: prospects for selection in a Mediterranean climate

The goals of the present study were to obtain a first estimate of intraspecific variability of carbon isotope discrimination (Δ) in safflower, a thistle-like herbaceous plant, and to determine the statistical relationship between Δ and grain yield as well as its components in a collection of 45 accessions of different origins. Grain yield and aboveground biomass, harvest index, average grain weight, and Δ (measured on the bulk leaf organic matter) were investigated in experimental field conditions. A large variability was noted for all traits but a principal component analysis (PCA) allowed to identify several homogeneous groups of accessions. Average grain yield per plant varied between 1 and 39 g. Δ varied between 21.3 and 25.2?‰, i.e. a large variation of 3.9?‰. In our experiment, the variation of Δ was not significantly related to that of grain yield in the whole accession sample. However, we found contrasting trends for this relation within accession groups. These initial results motivate further experiments to assess more in depth correlation between Δ and yield in safflower and are encouraging regarding the possibility of using Δ as an effective selection index in safflower to obtain genotypes that efficiently consume water. This study also highlighted one accession that combines the two characters required in the Mediterranean regions, i.e. high yield performance and high water-use efficiency.     

How elementary mercury reacts in the presence of halogen radicals and/or halogen anions: a DFT investigation

Reactions of elementary mercury in the gas phase (GEM) have been investigated at the DFT level in the presence of halogen radicals and/or halogen anions. In the presence of radicals, the formation of HgX(3)* and HgX(4)* is predicted to be favourable. Moreover, in the presence of anions, the free-radical liberation is enhanced from these two species allowing the presence of halogen free radicals even without the presence of light radiation. This enhancement is associated with the formation of HgX(3)(-), which is predicted to be the most stable species. In solution, redox chemistry can occur and transform GEM in the presence of X(2). The redox potentials of the couples HgX(2)/Hg for X=Cl, Br and I were calculated to be 0.52, 0.48 and 0.04 V, respectively. This study gives new opportunities to elucidate the environmental chemistry of Hg in the polar regions. In these areas GEM has a unique and fast reactivity due to a combination of factors such as the polar sunrise, the presence of halogenated radicals, snow and ice surfaces and cold temperatures. This reactivity, known as atmospheric mercury depletion events (AMDEs), leads to the deposition of significant amounts of Hg(2+) in these regions. The reaction pathways of AMDEs are as yet unknown and the DFT approach may contribute to their elucidation and to the proposal of new mechanisms. Additionally, this study introduces hypotheses concerning the reactivity of GEM inside snowpacks.     

Preparation and characterization of a microcrystalline non-heme FeIII(OOH) complex powder: EPR reinvestigation of FeIII(OOH) complexes-improvement of the perturbation equations for the g tensor of low-spin FeIII

The first example of a microcrystalline powder of a synthetic low-spin (LS) mononuclear Fe(III)(OOH) intermediate has been obtained by the precipitation of the [Fe(III)(L(5) (2))(OOH)](2+) complex at low temperature. The high purity of this thermally unstable powder is revealed by magnetic susceptibility measurements. EPR studies on this complex, in the solid state and also in frozen solution, are reported and reveal the coexistence of two related Fe(III)(OOH) species in both states. We also present a theoretical analysis of the g tensor for LS Fe(III) complexes, based on new perturbation equations. These simple equations provide distortion-energy parameters that are in good agreement with those obtained by a full-diagonalization calculation.     

Control of Immobilization and Hybridization on DNA Chips by Fluorescence Spectroscopy

A new methodology for the analysis of DNA polymorphisms has been developed using specific oligonucleotide strand arrays bound to a solid silicon support recovered by a thin layer of silica. Arrays of directly synthesized oligonucleotides covalently fixed on Si/SiO₂ wafers have been designed at the macroscopic scale. Using suitable nucleotide-labeled units, the fluorescence emission technique has been used as an experimental control of the molecular network bound to the support and as a method for analyzing the hybridizing abilities of the corresponding oligonucleotide array. Fluorescein has allowed us to control the molecular density of the DNA strand resulting from a complete synthetic growing process. A specific protocol using both complementary and noncomplementary units labeled with two probes, Cy3 and Cy5, was used to distinguish clearly nucleotide units fixed on the array either as hybridized sequences or by the unavoidable adsorption process. The present performance of this fluorescence detection procedure will now be used with a scanning fluorescence device to perform the analysis at the microscopic scale.     

Systems of coupled Poisson equations with critical growth in unbounded domains

We establish the existence of a nontrivial solution to systems of coupled Poisson equations with critical growth in unbounded domains. The proofs rely on a generalized linking theorem due to Krysewski and Szulkin, and on a concentration-compactness argument since the Palais-Smale condition fails at all critical levels.     

Reaction of naphthalene-2,3-dicarboxaldehyde with enkephalins for LC-fluorescence and lc-ms analysis: Conformational studies by molecular modeling and H/D Exchange mass spectrometry

A new labeling method compatible with both laser-induced fluorescence (LIF) and MS detection for enkephalins, which uses naphthalene-2,3-dicarboxaldehyde (NDA) and a new nucleophilic agent (N,N-dimethylaminoethanethiol) is described. When the derivative is separated via reverse phase HPLC and detected via MS, two different peaks with similar exact mass but different fluorescence and fragmentation properties are obtained. To interpret these results, molecular modeling and H/D exchange mass spectrometry studies were investigated to test the hypothesis that the peak obtained by LC/LIF/MS analysis depends on the site of protonation of the labeled enkephalins. The peptides labeled with NDA and N,N-dimethylaminoethanethiol were separated on a reverse phase C18 column with a gradient of aqueous 0.1% formic acid and acetonitrile. In mass spectrometry, two peaks are observed with the same exact mass for each molecule while only one peak is detected using fluorescence. Tandem mass spectrometry experiments of ion m/z 809.5 were performed on each chromatographic peak; the first peak (which is not observed by LIF detection) gives a fragment corresponding to the loss of the aminothiol side chain while no fragmentation is observed on the second peak, which was detected by fluorescence. The hypothesis is that each peak represents the labeled enkephalin with different sites of protonation. According to this hypothesis, three fundamental conformations that were closed to the unlabeled leucine-enkephalin were obtained by molecular modeling: a beta-turn like conformation with two hydrogen bonds, a 3(10)-helix with an H bond, and finally, the extended form without any intramolecular interactions. H/D exchange mass spectrometry experiments with D(2)O and d(2-)formic acid as eluent was used to determine which conformation is involved in each peak.