Biochemical characterization, molecular cloning and localization of a putative odorant-binding protein in the honey bee Apis mellifera L. (Hymenoptera: Apidea)

AbstractA honey bee antennal water-soluble protein, APS2, was purified and characterized as the first Hymenoptera putative odorant-binding protein. Comparison of its measured Mr (13 695.2±1.6) to that of the corresponding cDNA clone shows it does not undergo any post-translational modification other than a 19-residue signal peptide cleavage and formation of three disulfide bridges. These biochemical features are close to those of Lepidoptera odorant-binding proteins. In situ hybridization experiments demonstrated its specific expression in olfactory areas. Based on its higher expression in the worker than in the drone, ASP2 might be more involved in general odorant than in sex pheromone detection

Old divergence and restricted gene flow between torrent duck ( Merganetta armata ) subspecies in the Central and Southern Andes

Aim: To investigate the structure and rate of gene flow among populations of habitat‐specialized species to understand the ecological and evolutionary processes underpinning their population dynamics and historical demography, including speciation and extinction.Location: Peruvian and Argentine Andes.Taxon: Two subspecies of torrent duck (Merganetta armata).Methods: We sampled 156 individuals in Peru (M. a. leucogenis; Chillón River, n = 57 and Pachachaca River, n = 49) and Argentina (M. a. armata; Arroyo Grande River, n = 33 and Malargüe River, n = 17), and sequenced the mitochondrial DNA (mtDNA) control region to conduct coarse and fine‐scale demographic analyses of population structure. Additionally, to test for differences between subspecies, and across genetic markers with distinct inheritance patterns, a subset of individuals (Peru, n = 10 and Argentina, n = 9) was subjected to partial genome resequencing, obtaining 4,027 autosomal and 189 Z‐linked double‐digest restriction‐associated DNA sequences.Results: Haplotype and nucleotide diversities were higher in Peru than Argentinaacross all markers. Peruvian and Argentine subspecies showed concordant species‐level differences (ΦST mtDNA= 0.82;ΦST autosomal = 0.30;ΦST Z chromosome = 0.45),including no shared mtDNA haplotypes. Demographic parameters estimated formtDNA using IM and IMa2 analyses, and for autosomal markers using ∂a∂i (isolation‐with‐migration model), supported an old divergence (mtDNA = 600,000 years before present (ybp), 95% HPD range = 1.2 Mya to 200,000 ybp; and autosomal ∂a∂i = 782,490 ybp), between the two subspecies, characteristic of deeply divergedlineages. The populations were well‐differentiated in Argentina but moderately differentiated in Peru, with low unidirectional gene flow in each country.Main conclusions: We suggest that the South American Arid Diagonal was preexisting and remains a current phylogeographic barrier between the ranges of the two torrent duck subspecies, and the adult territoriality and breeding site fidelity to the rivers define their population structure.Fil: Alza, Luis. University of Alaska; Estados Unidos. University of Miami. Department of Biology; Estados Unidos. División de Ornitología. Centro de Ornitología y Diversidad; PerúFil: Lavretsky, Philip. University of Texas at El Paso; Estados UnidosFil: Peters, Jeffrey L.. Wright State University; Estados UnidosFil: Cerón, Gerardo. Universidad Nacional del Comahue. Centro Regional Universitario Bariloche. Departamento de Zoología. Laboratorio de Parasitología; ArgentinaFil: Smith, Matthew. University of Alaska; Estados UnidosFil: Kopuchian, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Centro de Ecología Aplicada del Litoral. Universidad Nacional del Nordeste. Centro de Ecología Aplicada del Litoral; Argentina. Museo Argentino de Ciencias Naturales, División Ornitología,; ArgentinaFil: Astié, Andrea Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Provincia de Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Universidad Nacional de Cuyo. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: McCracken, Kevin G.. División de Ornitología. Centro de Ornitología y Diversidad; Perú. University of Miami. Department of Biology; Estados Unidos. University of Miami; Estados Unidos. University of Alaska; Estados Unido

Enzyme-induced covalent modification of methionyl-tRNA synthetase from Bacillus stearothermophilus by methionyl-adenylate: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.

International audienceMethionyl-tRNA synthetase (MetRS) from Bacillus stearothermophilus was shown to undergo covalent methionylation by a donor methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by the enzyme itself. Covalent reaction of methionyl-adenylate with the synthetase or other proteins proceeds through the formation of an isopeptide bond between the carboxylate of the amino acid and the epsilon-NH2 group of lysyl residues. The stoichiometries of labeling, as followed by TCA precipitation, were 2.2 +/- 0.1 and 4.3 +/- 0.1 mol of [14C]Met incorporated by 1 mol of the monomeric MS534 and the native dimeric species of B. stearo methionyl-tRNA synthetase, respectively. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-261, -295, -301 and -528 (or -534) of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. By analogy with the 3D structure of the monomeric M547 species of E. coli methionyl-tRNA synthetase, lysines-261, -295, and -301 would be located in the catalytic crevice of the thermostable enzyme where methionine activation and transfer take place. It is proposed that, once activated by ATP, most of the methionine molecules react with the closest reactive lysyl residues

Resonance assignment, cysteine-pairing elucidation and secondary-structure determination of capsicein, an alpha-elicitin, by three-dimensional 1H NMR.

International audienceDifficulties encountered in the interpretation of two-dimensional NMR spectra of proteins exceeding roughly 100 amino acids, including resonance overlap and line broadening due to longer correlation times and/or aggregation phenomena, can be overcome by using three-dimensional 1H-NMR experiments. The improvement of spectral resolution using these experiments allows the size of molecules amenable to structure determination by NMR spectroscopy to be extended. A three-dimensional non-selective homonuclear Hartmann-Hahn/nuclear Overhauser effect spectroscopy experiment was performed on capsicein, a 10161-Da elicitin secreted by the Phytophthora capsici fungus. Sequential assignment and secondary structure determination is illustrated for beta-sheet, alpha-helix and loop structures by analysis of planar cross sections perpendicular to the omega 2 or omega 3 axis at the amide proton resonance frequencies. Cysteine pairing was established in the course of the investigation. The secondary structure topology of the molecule is composed of five helices and an antiparallel beta-sheet. Four of the helices compose two pairs running antiparallel while the last one is parallel to the beta-sheet

Three-dimensional solution structure of beta cryptogein, a beta elicitin secreted by a phytopathogenic fungus Phytophthora cryptogea.

Cryptogein belongs to a new family of 10-kDa proteins called elicitins. Elicitins are necrotic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The solution structure of beta cryptogein from Phytophthora cryptogea fungus was determined by using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. A set of 18 structures was calculated using 1360 NOE-derived distance restraints and 40 dihedral angle restraints obtained from 3JHNH alpha couplings. The RMS deviation from the mean structure is 0.87 +/- 0.14 A for backbone atoms and 1.34 +/- 0.14 A for all the non-hydrogen atoms of residues 2 to 98. The structure of beta cryptogein reveals a novel protein fold, with five helices and a double-stranded beta-sheet facing an omega-loop. One edge of the beta-sheet and the adjacent face of the omega-loop form a hydrophobic cavity. This cavity made of highly conserved residues represents a plausible binding site. Residue 13, which has been identified from directed mutagenesis and natural sequence comparison studies as a key amino acid involved in the differential control of necrosis, is surface exposed and could contribute to the binding to a ligand or a receptor. The solution structure is close to the X-ray structure, with slight differences lightly due to the crystal packing

1H and 15N resonance assignment and secondary structure of capsicein, an alpha-elicitin, determined by three-dimensional heteronuclear NMR.

International audienceThe backbone 1H and 15N resonance assignments and solution secondary structure determination of capsicein, a protein of 98 residues with a molecular mass of 10161 Da, are presented. Capsicein belongs to the elicitin family, elicitor molecules having toxic and signaling properties that are secreted by Phytophthora fungi, responsible for the incompatible hypersensitive reaction of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The protein was uniformly labeled with 15N to overcome spectral overlap of the proton resonances. A combination of 3D HOHAHA-HMQC and 3D NOESY-HMOC experiments allowed the identification of spin systems with through-bond correlations, which were in turn correlated by through-space connections. The sequential assignment was obtained for main- and side-chain resonances and led to the identification of all secondary structures. A 3D HMQC-NOESY-HMQC experiment was performed which characterized the NH(i)-NH(i+1) connections specific to alpha-helical structures. This proved particularly useful for the assignment of degenerate amide protons of successive residues in alpha-helical structures. The data show five alpha-helical regions comprising residues 5-18, 26-33, 44-58, 59-67, and 86-98 and a two-stranded antiparallel beta-sheet involving residues 70-75 and 80-85, packed around a hydrophobic core grouping all of the aromatic residues. The C-terminal secondary structure motifs of capsicein evoke phospholipase structural features, which suggests that elicitins might interact with the lipidic molecules of the plasma membrane

Separation, characterization and sexual heterogeneity of multiple putative odorant-binding proteins in the honeybee Apis mellifera L. (Hymenoptera: Apidea).

2 tables 5 graph.International audienceAccording to precise molar mass determined by mass spectrometry and N-terminal sequence, some 25 odorant-binding-like proteins were characterized from the antennae and legs of worker and drone honeybees. Antennal specific proteins, composed of six different molecules, were classified into three subclasses according to N-terminal sequence homology. The major sexual difference was shown to lie in the relative abundance of these antennal specific proteins and in the occurrence of a drone-specific isoform. At least 19 other related proteins were found to occur in antennae and legs, forming another class showing homology with insect OBP. Genotype comparison of two honeybee races revealed a variability limited to this second class. Provided that these odorant-binding-like proteins are indeed able to bind odorants or pheromones, the question of whether their peculiar multiplicity contributes to the remarkable capacity of the honeybee to discriminate among a wide range of odor molecules is raised

Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase.

International audienceValyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS