Electron Capture in Charge-Tagged Peptides. Evidence for the Role of Excited Electronic States

International audienceElectron capture dissociation (ECD) was studied with doubly charged dipeptide ions that were tagged with fixed-charge tris-(2,4,6-trimethoxyphenyl)phosphonium-methylenecarboxamido (TMPP-ac) groups. Dipeptides GK, KG, AK, KA, and GR were each selectively tagged with one TMPP-ac group at the N-terminal amino group while the other charge was introduced by protonation at the lysine or arginine side-chain groups to give (TMPP-ac-peptide + H)2+ ions by electrospray ionization. Doubly tagged peptide derivatives were also prepared from GK, KG, AK, and KA in which the fixed-charge TMPP-ac groups were attached to the N-terminal and lysine side-chain amino groups to give (TMPP-ac-peptide-ac-TMPP)2+ dications by electrospray. ECD of (TMPP-ac-peptide + H)2+ resulted in 72% to 84% conversion to singly charged dissociation products while no intact charge-reduced (TMPP-ac-dipeptide + H)+• ions were detected. The dissociations involved loss of H, formation of (TMPP + H)+, and N–C(alpha) bond cleavages giving TMPP-CH2CONH2+ (c0) and c1 fragments. In contrast, ECD of (TMPP-ac-peptide-ac-TMPP)2+ resulted in 31% to 40% conversion to dissociation products due to loss of neutral TMPP molecules and 2,4,6-trimethoxyphenyl radicals. No peptide backbone cleavages were observed for the doubly tagged peptide ions. Ab initio and density functional theory calculations for (Ph3P-ac-GK + H)2+ and (H3P-ac-GK + H)2+ analogs indicated that the doubly charged ions contained the lysine side-chain NH3+ group internally solvated by the COOH group. The distance between the charge-carrying phosphonium and ammonium atoms was calculated to be 13.1-13.2 Å in the most stable dication conformers. The intrinsic recombination energies of the TMPP+-ac and (GK + H)+ moieties, 2.7 and 3.15 eV, respectively, indicated that upon electron capture the ground electronic states of the (TMPP-ac-peptide + H)+• ions retained the charge in the TMPP group. Ground electronic state (TMPP-ac-GK + H)+• ions were calculated to spontaneously isomerize by lysine H-atom transfer to the COOH group to form dihydroxycarbinyl radical intermediates with the retention of the charged TMPP group. These can trigger cleavages of the adjacent N–C(alpha) bonds to give rise to the c1 fragment ions. However, the calculated transition-state energies for GK and GGK models suggested that the ground-state potential energy surface was not favorable for the formation of the abundant c0 fragment ions. This pointed to the involvement of excited electronic states according to the Utah-Washington mechanism of ECD

Differential regulation of gene products in newly synthesized Brassica napus allotetraploids is not related to protein function nor subcellular localization

BACKGROUND: Allopolyploidy is a preeminent process in plant evolution that results from the merger of distinct genomes in a common nucleus via inter-specific hybridization. Allopolyploid formation is usually related to genome-wide structural and functional changes though the underlying mechanisms operating during this "genomic shock" still remain poorly known. The aim of the present study was to investigate the modifications occurring at the proteomic level following an allopolyploidization event and to determine whether these changes are related to functional properties of the proteins. In a previous report, we applied comparative proteomics to synthetic amphiploids of Brassica napus and to its diploid progenitors B. rapa and B. oleracea. Although several hundred polypeptides displayed additivity (i.e. mid-parent values) in the amphiploids, many of them showed non-additivity. Here, we report the in silico functional characterization of the "non-additive" proteins (the ones with a non-additive pattern of regulation) in synthetic B. napus. RESULTS: The complete set of non-additive proteins (335 in the stem and 205 in the root), as well as a subset of additive polypeptides (200 per organ), was identified by mass spectrometry. Several protein isoforms were found, and most of them (~55%) displayed "different" or "opposite" patterns of regulation in the amphiploids, i.e. isoforms of the same protein showing both up-regulation and down-regulation in the synthetic B. napus compared to the mid-parent value. Components of protein complexes were identified of which ~50% also displayed "different" or "opposite" patterns of regulation in the allotetraploids. In silico functional categorization of the identified proteins was carried out, and showed that neither functional category nor metabolic pathway were systematically affected by non-additivity in the synthetic amphiploids. In addition, no subcellular compartment was found to be over- or under-represented among the proteins displaying non-additive values in the allopolyploids. CONCLUSION: Protein identification showed that functionally related polypeptides (isoforms and complex subunits) could be differentially regulated in synthetic B. napus in comparison to its diploid progenitors while such proteins are usually expected to display co-regulation. The genetic redundancy within an allopolyploid could explain why functionally related proteins could display imbalanced levels of expression. No functional category, no metabolic pathway and no subcellular localization was found to be over- or under-represented within non-additive polypeptides, suggesting that the differential regulation of gene products was not related to functional properties of the proteins. Thus, at the protein level, there is no evidence for the "genomic shock" expected in neo-polyploids and the overall topology of protein networks and metabolic pathways is conserved in synthetic allotetraploids of B. napus in comparison to its diploid progenitors B. rapa and B. oleracea

Solid-Phase Microextraction and Cuticular Hydrocarbon Differences Related to Reproductive Activity in Queenless Ant Dinoponera quadriceps

International audienc

Le dosage des acides gras phospholipidiques des sols : limites d’interprétations

Le dosage des acides gras phospholipidiques des sols : limites d’interprétations. 1er Symposium de Chimie et Biologie Analytique

Efficiency of spraying mountain pine cones with oleoresin of Swiss stone pine cones to prevent insect attack

31 ref.International audienceThe difference in cone colonization by specialized insects that had been observed between Swiss stone pine (Pinus cembra L.) and mountain pine (P. uncinata Ram.) prompted us to test whether volatiles and oleoresin of Swiss stone pine cones may serve as a barrier for insect attack. The volatiles emitted by cones of both pine species were sampled using headspace techniques in the French Alps. The cone volatiles of both species essentially consisted of seven similar monoterpenes, but the terpene profiles markedly differed between species. α-Pinene (67% of the total), β-pinene, and limonene + β-phellandrene were dominant in the cone volatiles of Swiss stone pine, while the cone emissions of mountain pine showed no dominant terpenoid. The cone oleoresin of P. cembra significantly differed from cone volatiles by a lower level of α-pinene and higher levels of β-pinene and limonene + β-phellandrene. Field sprays of mountain pine cones with oleoresin extracts of Swiss stone pine cones significantly reduced the overall damage of specialized cone insects. None of the cones sprayed with oleoresin were attacked, whereas 11% and 31% of the unsprayed control cones were damaged by insects. Specific cone damage due to a cone weevil, Pissodes validirostris Gyll. (Coleoptera: Curculionidae), and a cone pyralid, Dioryctria mutatella Fuchs (Lepidoptera: Pyralidae), were significantly decreased in one year. The volatile profile of the sprayed cones differed markedly from that of unsprayed mountain pine cones, especially in the higher level of myrcene. However, the volatile profile of the sprayed cones also differed from that of the oleoresin extract used for spraying. The role of monoterpenes in protecting the sprayed cones from insect attack is discussed

Gas-phase basicity of methionine.

International audienceProton affinity and protonation entropy of methionine (Met) were determined by the extended kinetic method from ESI-Q-TOF tandem mass spectrometry experiments. The values, PA(Met) = 937.5 +/- 2.9 kJ mol(-1) and Delta(p)S degrees (Met) = - 22 +/- 5 J mol(-1) K(-1), lead to gas-phase basicity GB(Met) = 898.2 +/- 3.2 kJ.mol(-1). Quantum chemical calculations using density functional theory confirm that the proton affinity of Met is indeed in the 940 kJ mol(-1) range and that a significant entropy loss, of at least - 25 J mol(-1) K(-1), occurs upon protonation. This last point is evidenced here for the first time and suggests revision of the tabulated protonation thermochemistry of Met. A comparison with previous experimental data allows us to propose the following evaluated thermochemical values: PA(Met) = 943 +/- 4 kJ mol(-1) and Delta(p)S degrees (Met) = - 35 +/- 15 J mol(-1) K(-1) and GB(Met) = 900 +/- 2 kJ mol(-1)

EF-Tu from the enacyloxin producing Frateuria W-315 strain: Structure/activity relationship and antibiotic resistance

International audienceIn this report, we have demonstrated that the poly(U)-dependent poly(Phe) synthesis activity of elon-gator factor Tu (EF-Tu) from the enacyloxin producing strain Frateuria sp. W-315 is inhibited by the antibiotic similarly to that of Escherichia coli EF-Tu. The inhibitory effect of enacyloxin observed in a purified system was the same as that obtained with an S30 extract from E. coli or Frateuria sp. W-315, respectively, suggesting that antibiotic resistance of enacyloxin producing Frateuria sp. W-315 is not due neither to EF-Tu nor to other components of the translation machinery but to a still unknown mechanism. The EF-Tu gene, as PCR amplified from Frateuria W-315 genomic DNA and sequenced represented an ORF of 1191 nucleotides corresponding to 396 amino acids. This protein is larger than the product of tufA from E. coli by only two amino acid residues. Alignment of the amino acid sequence of EF-Tu from E. coli with those of Frateuria and Ralstonia solanacearum indicates on average 80% identical amino acid residues and 9.7% conservative replacements between EF-Tu Frateuria and EF-Tu E. coli, on one hand, and 97% identity and 1.7% conservative replacement between EF-Tu Frateuria and EF-Tu Ralstonia sol-anacearum, on the other hand. These strong primary structure similarities between EF-Tu from different origins are consistent with the fact that this factor is essential for the translation process in all kingdoms of life. Comparison of the effects of antibiotics on EF-Tu Frateuria and EF-Tu E. coli revealed that enacyloxin, kirromycin and pulvomycin exert a stronger stimulation of the GDP dissociation rate on EF-Tu Frateuria, while the effects of the antibiotics on the GDP association rate were comparable for the two EF-Tu species. Different mutants of EF-Tu E. coli were constructed with the help of site directed muta-genesis by changing one or several residues of EF-Tu E. coli by the corresponding residues of EF-Tu Frateuria. The single A45K substitution did not modify the intrinsic GTPase activity of EF-Tu E. coli. In contrast, a 2e3 fold stimulation of the intrinsic GTPase activity was observed with the single A42E, F46Y, Q48E and the double F46Y/Q48E substitution. Finally, up to a 7 fold stimulation was observed with the quadruple substitution (mutant A42E/A45K/F46Y/Q48E