Fluorescence Quenching as a Tool to Investigate Quinolone Antibiotic Interactions with Bacterial Protein OmpF |
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Authors: | Patrícia Neves Isabel Sousa Mathias Winterhalter Paula Gameiro |
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Institution: | (1) Requimte, Faculdade de Ciências, Universidade do Porto, Rua Campo Alegre, 4169-007 Porto, Portugal;(2) Jacobs University Bremen, 28726 Bremen, Germany |
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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 (Trp214, Trp61), 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 Trp61 and iodide Trp214. Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic
acid interacting near Trp214 and moxifloxacin near Trp61. 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. |
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Keywords: | Spectrofluorescence Biophysics Structure of membrane protein Fluorescent probe |
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