A high-throughput screen for identifying transmembrane pore-forming peptides.
We have developed a visual microwell plate assay for rapid, high-throughput screening for membrane-disrupting molecules such as de novo designed pore formers, antibiotic peptides, bacterial toxins, and lipases. The detectability is based on the strong fluorescence emission of the lanthanide metal terbium(III) (Tb(3+)) when it interacts with the aromatic chelator dipicolinic acid (DPA). While Tb(3+) is not strongly fluorescent alone, the binary complex emits bright green fluorescence when irradiated with uv light. For the microwell plate assay, we prepared unilamellar phospholipid vesicles that had either Tb(3+) or DPA entrapped and the opposite molecule in the external solution. Disruption of the membranes allows the Tb(3+)/DPA complex to form, giving rise to a visibly fluorescent solution. In plates with 20-microl wells, the lower limit of visual detectability of the Tb(3+)/DPA complex in solution was about 2.5 microM. The lower limit of detectability using vesicles with entrapped Tb(3+) or DPA was about 50 microM phospholipid. We show that the membrane-disrupting effect of as little as 0.25 microM or 5 pmol of the pore-forming, antibiotic peptide alamethicin can be detected visually with this system. This sensitive, high-throughput assay is readily automatable and makes possible the visual screening of combinatorial peptide libraries for members that permeabilize lipid bilayer membranes.
Rausch
J M
JM
Department of Biochemistry SL43, Tulane University Health Sciences Center, New Orleans, Louisiana 70112-2699, USA.
Wimley
W C
WC
D000408:Alamethicin / Q000032:analysis / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000032:analysis / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D007476:Ionophores / Q000032:analysis / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000032:analysis* / Q000378:metabolism*; D010539:Permeability; D010848:Picolinic Acids / Q000378:metabolism; D018272:Porins / Q000032:analysis* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013725:Terbium / Q000378:metabolism
D000900:Anti-Bacterial Agents; D007476:Ionophores; D008051:Lipid Bilayers; D008081:Liposomes; D010455:Peptides; D010848:Picolinic Acids; D018272:Porins; D013725:Terbium; D000408:Alamethicin; C004860:dipicolinic acid