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Etude structurale et fonctionnelle de peptides lasso à l'aide de méthodes spectroscopiques

Abstract : Lasso peptides are ribosomally-synthesized and post-translationnally modified peptides produced by bacteria, sharing a knotted topology. They present a N-terminal macrolactam ring and the C-terminal tail is threaded through this ring. Strong sterical constraints and/or disulfide bridges stabilize this scaffold. Due to there biological activities (as receptor antagonists, enzyme inhibitors which confer them antiviral or antibacterial activity) and the high stability of there "lasso" structure, they present a great biotechnical interest. This PhD work participates to the comprehension of the sequence determinants governing the topology and the activity of these peptides, using the development of spectroscopic methods. Structure/activity relationships of microcin J25 (MccJ25), an antibacterial lasso peptide produced by Escherichia coli, were examined. MccJ25 variants were generated with a site-directed mutagenesis onto the gene encoding the precursor. The topology of the variants (lasso or cyclic-branched) was determined using LC- MS/MS, carboxypeptidase Y digestion, and in some case NMR. The antibacterial activity on Salmonella enterica was measured. Determinants governing the sterical trapping of the C-terminal tail were identified. Residues under the ring (Tyr20, Gly21) and the size of the rign are critical for the topology and the antibacterial activity, whereas the reduction of the loop Tyr9-Ser18 above the ring or the elongation of the C-terminal tail under the cycle maintain the lasso topology but decrease or abolish the activity.We isolated and characterized a new lasso peptide from Streptomyces sviceus and predicted by genome-mining, sviceucin. The tridimensional structure of the peptide was determined by solution NMR. This peptide presents a C-terminal tail threaded through a nine-residues macrolactam ring. Two disulfide bridges between the ring and the tail, Cys1/Cys13 and Cys7/Cys19, stabilize the structure. This new peptide is highly similar to other antimicrobial and antiviral lasso peptides, as RP-71955. He presents an antibacterial activity against Gram-positive bacteria. Finally, a spectroscopic study of several lasso peptides permits us to describe specific signals of structural regions of lasso peptides. The dynamic study of MccJ25 by 15N spin relaxation shows an inhomogeneity of internal mobility of residues according to their position on the "lasso" topology. Moreover, the study by circular dichroism of MccJ25, sviceucin and capistruin (lasso peptide from Burkholderia thailendensis) reveals that some lasso peptides present a 220 nm positive band, specific of the lasso structure
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Rémi Ducasse. Etude structurale et fonctionnelle de peptides lasso à l'aide de méthodes spectroscopiques. Biochimie, Biologie Moléculaire. Université Pierre et Marie Curie - Paris VI, 2012. Français. ⟨NNT : 2012PAO66389⟩. ⟨tel-00828620⟩

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