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Updated: Jun 26, 2026
Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes
Published on: March 2, 2020
Fluorescence quenching as a tool to investigate quinolone antibiotic interactions with bacterial protein OmpF
Patrícia Neves1, Isabel Sousa, Mathias Winterhalter
1Universidade do Porto, Rua Campo Alegre, Portugal.
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.
Insights
Outer membrane porin OmpF facilitates antibiotic uptake in gram-negative bacteria. Fluorescence quenching experiments identified specific tryptophan residues involved in quinolone antibiotic binding, revealing distinct interaction sites for nalidixic acid and moxifloxacin.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- The outer membrane porin OmpF is crucial for antibiotic entry into gram-negative bacteria.
- Understanding antibiotic binding sites on OmpF is key to elucidating mechanisms of action and bacterial resistance.
- The specific molecular sites of antibiotic interaction with OmpF remain largely uncharacterized.
Purpose of the Study:
- To identify the molecular binding sites of quinolone antibiotics on the OmpF porin.
- To investigate the role of specific tryptophan residues in OmpF-antibiotic interactions.
- To explore protein conformational changes upon antibiotic binding.
Main Methods:
- Utilized intrinsic fluorescence of OmpF, specifically tryptophan residues (Trp(61) and Trp(214)).
- Employed fluorescence quenching experiments with acrylamide and iodide to probe tryptophan accessibility.
- Analyzed quinolone antibiotic (nalidixic acid, moxifloxacin) interactions with reconstituted OmpF.
Main Results:
- Acrylamide preferentially quenched Trp(61), while iodide quenched Trp(214) in OmpF.
- Nalidixic acid was found to interact near Trp(214).
- Moxifloxacin was observed to interact near Trp(61).
- Conformational changes in OmpF were detected upon interaction with different quinolones.
Conclusions:
- Distinct binding sites on OmpF exist for different quinolone antibiotics.
- Nalidixic acid and moxifloxacin interact with specific tryptophan residues (Trp(214) and Trp(61), respectively).
- OmpF undergoes conformational reorganization to accommodate quinolone binding, providing insights into antibiotic interaction and resistance.
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