title: High-throughput and facile assay of antimicrobial peptides using pH-controlled fluorescence resonance energy transfer.
abstract: Amphipathic antimicrobial peptides can destroy bacteria cells by inducing membrane permeabilization, forming one strategy for innate defense by various organisms. However, although the antimicrobial peptides are considered a promising alternative for use against multidrug-resistant bacteria, large-scale screening of potential candidate antimicrobial peptides will require a simple, rapid assay for antimicrobial activity. Here, we describe a novel fluorescence resonance energy transfer (FRET)-based assay system for antimicrobial peptides which takes advantage of pH-related changes in FRET efficiency due to the instability of enhanced yellow fluorescent protein versus the stability of enhanced cyan fluorescent protein in a reduced-pH environment. We successfully showed that quantification of antimicrobial activity is possible through a difference of FRET efficiency between ECFP-EYFP fusion molecules released from disrupted Escherichia coli in an extracellular environment (pH 6) and those retained in an intracellular environment (pH approximately 7). Thus, we herein suggest a new simple, effective, and efficient pH-controlled FRET-based antimicrobial peptide screening method applicable to high-throughput screening of candidate peptide libraries.
authors.affiliation: Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Korea.
authors.forename: Young Soo
authors.initials: YS
authors.lastname: Kim
authors.forename: Hyung Joon
authors.initials: HJ
authors.lastname: Cha
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D054806:Magainins; D008826:Microbial Sensitivity Tests / Q000379:methods*; D029867:Xenopus Proteins / Q000494:pharmacology
chemical_list: D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D054806:Magainins; D029867:Xenopus Proteins; C054562:yellow fluorescent protein, Bacteria; C053301:magainin 2 peptide, Xenopus; D049452:Green Fluorescent Proteins