A low-temperature heteronuclear NMR study of two exchanging conformations of metal-bound pyoverdin PaA fromPseudomonas aeruginosa

Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdin PaA, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, D-Ser–L-Arg–D-Ser–L-FoOHOrn–(L-Lys–L-FoOHOrn–L-Thr–L-Thr) (FoOHOrn: δN-formyl-δN-hydroxyornithine), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the L-Lys side chain. Ferric iron is chelated by the catechol group on the chromophore and the two hydroxyornithine side chains. In aqueous solution, the 1H-NMR spectrum of pyoverdin PaA–Ga(III), in which Ga(III) is used instead of Fe(III) for spectroscopic purposes, showed clear evidence of exchange broadening, preventing further structural characterization. The use of cryo-solvents allowed measurements to be made at temperatures as low as 253 K where two distinct conformations with roughly equivalent populations could be observed. 13C and 15N labeling of pyoverdin PaA enabled complete assignment of both forms of pyoverdin PaA–Ga(III) at 253 and 267 K, using triple-resonance multidimensional NMR experiments commonly applied to doubly labeled proteins. © 2005 Wiley Periodicals, Inc. Biopolymers 79: 139–149, 2005 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com