Cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC form the bacteriocin colicin V (ColV) secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter with nucleotide-binding motifs in the C-terminal domain (CTD). To study the role of CvaB-CTD in the ColV secretion, a truncated construct of this domain was made and over-expressed. Different forms of CvaB-CTD were obtained during purification, and were identified as monomer, dimer, and oligomer on gel filtration. Nucleotide binding was shown critical for the CvaB-CTD dimerization: oligomers could be converted into dimers by nucleotide bindings; the removal of nucleotide from dimers resulted in transient monomers followed by CTD oligomerization and aggregation; no dimer form could be cross-linked from the nucleotide-binding deficient mutant D654H. The spatial proximity of the Walker A site and ABC signature motif in CTD dimer was identified through disulfide cross-linking of mixed CvaB-CTD with mutants A530C and L630C, while mutations did not dimerize individually. Those results indicated that the CvaB-CTD formed a nucleotide-dependent head-to-tail dimer. Molecular basis of differential nucleotide bindings was also studied through bioinformatics prediction and biochemical verification. Through sequence alignment and homology modeling with bound ATP or GTP, it was found that the Ser503 and Gln504 on aromatic stacking region (Y501DSQ-loop) of CvaB-CTD provided two additional hydrogen-bonds to GTP, but not to ATP. Site-directed mutations of the S503A and/or Q504L were designed based on the model. While site-directed mutagenesis studies of Walker A&B sites or the ABC signature motif affected little on the GTP-binding preference, the double mutation (S503A/Q504L) on the Y501DSQ-loop increased both ATP-binding and ATPase activity at low temperatures. The double mutant showed slight decrease of GTP-binding and about 10-fold increase of the ATP/GTP-binding ratio. Similar temperature sensitivity in nucleotide-binding and activity assays were identified in the double mutant at the same time. Mutations on the Y501DSQ-loop did not affect the ColV secretion level in vivo. Together, the Y501DSQ-loop is structurally involved in the differential binding of GTP over ATP.

Ce travail a pour objectif d’augmenter la diversité des foldamères par le développement d’une nouvelle classe d’architectures abiotiques mimant le repliement des feuillets β protéiques. La stratégie employée repose sur des processus de structuration qui diffèrent de ceux observés au sein du vivant. Les deux éléments essentiels des systèmes naturels, à savoir la boucle courte et flexible d’acides aminés et les brins β liés entre eux par des liaisons hydrogènes, sont ici substitués respectivement par un coude rigide formé de noyaux aromatiques et par des oligoarylamides plans interagissant par empilement aromatique.Ces objets ont été conçus pour adopter des structures repliées caractérisables en solution par spectroscopie RMN et à l’état solide par diffraction des rayons X. Dans une première partie, l’optimisation du pseudo-coude β et de la séquence des brins, ainsi que l’exploration des premières architectures en feuillet seront étudiées à travers la conception, la synthèse et les études structurales de ces oligoamides et oligoamines aromatiques. Dans une seconde partie, le concept mis en oeuvre sera étendu à la synthèse de foldamères plus élaborés à brins courbés, ouvrant ainsi des perspectives intéressantes vers l’obtention d’architectures toujours plus complexes. / The purpose of this work is to expand foldamer diversity by developing a novel class of abiotic β-sheet-like architectures. Our strategy uses inter-strand π-π aromatic stacking between sequences of aromatic oligoamides and oligoamines to mimic the natural stabilization of β-strands, which occurs through a network of regularly spaced hydrogen bonds. These oligamide and oligoamine sequences are connected by a rigid U-shaped moiety that creates a turn and initiates strand formation.These molecules have been designed to adopt compact folded structures that can be studied in solution by NMR spectroscopy and in the solid state by X-ray crystallography. In the first part of this dissertation, we report our stepwise approach in the development of β-sheet-like aromatic amide-based foldamers: from the optimization of the design elements and the use of macrocycles, to the synthesis of multi-turn structures. In the second part, the concept will be extented to the synthesis of more elaborate curving strand β-sheet-like foldamers, opening up new perspectives for more complex architectures.