Gramicidin-based fluorescence assay; for determining small molecules potential for modifying lipid bilayer properties

Area of Science:

• Biophysics
• Membrane Biology
• Pharmacology

Background:

• Amphiphilic drugs can alter lipid bilayer properties by adsorbing at the membrane interface.
• Changes in bilayer properties can affect membrane protein function, leading to potential off-target drug effects.
• Linear gramicidin channels are sensitive to their membrane environment, serving as effective probes for bilayer properties.

Purpose of the Study:

• To develop and validate a fluorescence-based assay for detecting changes in lipid bilayer properties.
• To utilize gramicidin channels as probes in a fluorescence assay for high-throughput screening.
• To assess the utility of this assay for identifying amphiphiles with potential off-target effects.

Main Methods:

• Developed a fluorescence assay using gramicidin channels incorporated into large unilamellar vesicles.
• Employed a fluorescence indicator/quencher pair (8-aminonaphthalene-1,3,6-trisulfonate/Tl+) to monitor molecular entry.
• Measured the time-course of fluorescence quenching as Tl+ permeated through gramicidin channels.

Main Results:

• The fluorescence assay successfully detected changes in lipid bilayer properties induced by small molecules.
• Results obtained using the fluorescence assay showed good agreement with previous electrophysiological measurements.
• The assay is scalable and suitable for both mechanistic studies and high-throughput screening.

Conclusions:

• A novel, scalable fluorescence assay using gramicidin channels can effectively detect bilayer-perturbing effects of small molecules.
• This assay provides a valuable tool for identifying potential off-target drug effects and understanding drug-membrane interactions.
• The fluorescence method complements existing electrophysiological techniques for membrane biophysics research.