Fluorescence Quenching as a Tool to Investigate Quinolone Antibiotic Interactions with Bacterial Protein OmpF

authors	Neves, P; Sousa, I; Winterhalter, M; Gameiro, P
journal	JOURNAL OF MEMBRANE BIOLOGY
author keywords	Spectrofluorescence; Biophysics; Structure of membrane protein; Fluorescent probe
keywords	ESCHERICHIA-COLI; STAPHYLOCOCCUS-AUREUS; PSEUDOMONAS-AERUGINOSA; PORIN; ACCUMULATION; FLUOROQUINOLONES; CIPROFLOXACIN; MOXIFLOXACIN; PERMEATION; LIPOSOMES
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.
publisher	SPRINGER
issn	0022-2631
year published	2009
volume	227
issue	3
beginning page	133
ending page	140
digital object identifier (doi)	10.1007/s00232-008-9152-2
web of science category	Biochemistry & Molecular Biology; Cell Biology; Physiology
subject category	Biochemistry & Molecular Biology; Cell Biology; Physiology
unique article identifier	WOS:000263916400004