Terbium, a fluorescent probe for investigation of siderophore pyochelin interactions with its outer membrane transporter FptA |
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Authors: | Yang Binsheng Hoegy Françoise Mislin Gaëtan L A Mesini Philippe J Schalk Isabelle J |
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Institution: | a Institute of Molecular Science, Shanxi University, Taiyuan, PR Chinab UMR7242, Université de Strasbourg-CNRS, ESBS, Blvd Sébastien Brant, F-67413 Illkirch, Strasbourg, Francec Institut Charles Sadron, Université de Strasbourg-CNRS, 23 rue du Loess, 67034 Strasbourg Cedex 2, France |
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Abstract: | Pyochelin (Pch) is a siderophore and FptA is its outer membrane transporter produced by Pseudomonas aeruginosa to import iron. The fluorescence of the element terbium is affected by coordinated ligands and it can therefore be used as a probe to investigate the pyochelin-iron uptake pathway in P. aeruginosa. At pH 8.0, terbium fluorescence is greatly enhanced in the presence of pyochelin indicating chelation of the metal by the siderophore. Titration curves showed a 2:1 (Pch:Tb3+) stoichiometry and an affinity of K =( 2 ± - 1 )× 1011 M− 2 was determined. Pch-Tb interaction with the transporter FptA could be followed in vitro and in vivo in P. aeruginosa cells, by Fluorescence Resonance Energy Transfer (FRET) between three partners: the tryptophans of FptA (donor), Pch (acceptor for the Trps and donor for Tb3+) and Tb3+ (acceptor). Pch-Tb binds to the Pch-Fe outer membrane transporter FptA with a dissociation constant (Kd) of 4.6 μM. This three-partner FRET is a potentially valuable tool for investigation of the interactions between FptA and its siderophore Pch. |
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Keywords: | Terbium Pyochelin Siderophore Pseudomonas aeruginosa Metal traffic Fluorescence Resonance Energy Transfer |
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