High Throughput Screen of Combinatorial Peptide Library for Gain-of-Function and Loss-of-Function Changes to Melittin

Abstract

Melittin, the main peptide component of European Honey Bee venom, is an amphipathic, 26-amino acid peptide that lyses bacterial and mammalian cells by forming transmembrane pores. Our research focuses on the various mechanisms of peptide permeation of membranes. We have designed orthogonal, fluorescence-based assays to characterize long-lived pore-forming peptides such as melittin. In these assays, peptides are incubated overnight with vesicles containing dye-labeled lipids and entrapped terbium. In the first measurement, the sum of the lytic activity is determined by measuring the terbium released from the vesicles. In the second measurement, we add a non membrane-permeable quencher of dye-labeled lipids. Using these assays we have observed a significant difference between melittin and other lytic peptides, such as alamethicin. Even at very high lipid concentrations (peptide:lipid < 1:2000) alamethicin forms long-lived pores, which release ∼100% of entrapped contents and give the quencher 100% access to the inside of a vesicle after overnight incubation. Melittin's activity diminishes significantly at lipid concentrations larger than P:L = 1:500. To learn what factors modulate melittin's activity, we have designed a 7,776-member, melittin-based combinatorial peptide library in which we vary critical residues in its natural sequence. We also incorporate self-associating motifs that are found in alpha-helical membrane proteins. Library members were screened using the orthogonal assays at very high and very low stringency. Selected positives from the highly stringent assay (i.e. gain in activity sequences) show a high frequency of alanine substitutions at specific polar and basic residues. Selected negatives from the low-stringency assay (i.e. loss of activity sequences) show two key nonpolar-to-glycine replacements as well as a substitution of the proline residue. Selected peptides from both screens have been contrasted to melittin's activity by using biophysical techniques, antimicrobial and hemolytic assays.