title: Fluorescence quenching as a tool to investigate quinolone antibiotic interactions with bacterial protein OmpF.
abstract: The outer membrane porin OmpF is an important protein for the uptake of antibiotics through the outer membrane of gram-negative bacteria; however, the possible binding sites involved in this uptake are still not recognized. Determination, at the molecular level, of the possible sites of antibiotic interaction is very important, not only to understand their mechanism of action but also to unravel bacterial resistance. Due to the intrinsic OmpF fluorescence, attributed mainly to its tryptophans (Trp(214), Trp(61)), quenching experiments were used to assess the site(s) of interaction of some quinolone antibiotics. OmpF was reconstituted in different organized structures, and the fluorescence quenching results, in the presence of two quenching agents, acrylamide and iodide, certified that acrylamide quenches Trp(61) and iodide Trp(214). Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic acid interacting near Trp(214) and moxifloxacin near Trp(61). These studies, based on straightforward and quick procedures, show the existence of conformational changes in the protein in order to adapt to the different organized structures and to interact with the quinolones. The extent of reorganization of the protein in the presence of the different quinolones allowed an estimate on the sites of protein/quinolone interaction.
authors.affiliation: Universidade do Porto, Rua Campo Alegre, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Neves
authors.forename: Isabel
authors.initials: I
authors.lastname: Sousa
authors.forename: Mathias
authors.initials: M
authors.lastname: Winterhalter
authors.forename: Paula
authors.initials: P
authors.lastname: Gameiro
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001665:Binding Sites; D018272:Porins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D015363:Quinolones / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
chemical_list: D000900:Anti-Bacterial Agents; C109237:OmpF protein; D018272:Porins; D015363:Quinolones