A high-throughput screen for identifying transmembrane pore-forming peptides

被引:28
作者
Rausch, JM [1 ]
Wimley, WC [1 ]
机构
[1] Tulane Univ, Hlth Sci Ctr, Dept Biochem SL43, New Orleans, LA 70112 USA
关键词
terbium; Tb3+; dipicolinic acid; DPA; combinatorial; alamethicin;
D O I
10.1006/abio.2001.5137
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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) (Tb3+) when it interacts with the aromatic chelator dipicolinic acid (DPA). While Tb3+ 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 Tb3+ or DPA entrapped and the opposite molecule in the external solution. Disruption of the membranes allows the Tb3+/DPA complex to form, giving rise to a visibly fluorescent solution. In plates with 20-mul wells, the lower limit of visual detectability of the Tb3+/DPA complex in solution was about 2.5 muM. The lower limit of detectability using vesicles with entrapped Tb3+ or DPA was about 50 muM phospholipid. We show that the membrane-disrupting effect of as Little as 0.25 muM or 5 pmol of the pore-forming, antibiotic peptide alamethicin dan 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. (C) 2001 Academic Press.
引用
收藏
页码:258 / 263
页数:6
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