Design and characterization of pore-forming peptides from a combinatorial library
Description
Membrane pores are essential structures for cells. These proteins control the flow of material between the external environment and the cytoplasm by permeating the hydrophobic barrier of the lipid bilayer membranes. Small, amphipathic, cationic peptides produced by a variety of organisms have been shown to permeabilize lipid membranes as transmembrane pores. These peptides participate in critical functions of host defense by targeting the anionic, outer surfaces of bacterial membranes. Upon partitioning into membranes, these peptides assemble to form pores, inducing unregulated leakage of cellular contents. The design and control of such peptides would be an advance in the development of novel antibiotics. These peptides must satisfy hydrogen-bonding requirements by forming secondary structures upon partitioning. Peptides adopting beta-hairpin conformations may be effective mimics of the beta-barrel pore structure. A combinatorial library of nearly 10,000 member peptides designed to form beta-hairpins was synthesized and tested using a visual fluorescent assay for detection of pore formation in lipid vesicles. Peptides selected from this library for their ability to permeabilize lipid vesicles at peptide to lipid ratios of 1:500 in this screening assay were sequenced to identify amino acid substitutions in combinatorial sites. Leakage competent peptides were synthesized from these sequences and characterized spectroscopically for their peptide-peptide and peptide-lipid interactions. The peptides were found to induce pore formation through a carpet model mechanism by these studies. Antibacterial assays indicated that these peptides are capable of killing both gram-negative and gram-positive species at low muM concentrations