A Novel Functional Class of Pore-Forming Peptides

Abstract

Peptides that self-assemble into membrane spanning pores, at low concentration, to allow the passage of macromolecules would be beneficial in multiple areas in biotechnology, including biosensor design and drug delivery. However, there are few, if any, natural or designed peptides have this property. Here we show that the 26-residue peptide “MelP5”, a gain-of-function variant of the bee venom lytic peptide melittin, identified in a high-throughput screen for small molecule leakage, enables the passage of macromolecules across bilayers. In surface supported bilayers, MelP5 forms unusually high conductance, equilibrium pores at low peptide concentration, and also increases the capacitance of the supported bilayers, suggesting that it forms large pores that affect a significant fraction of the membrane surface. The increase in bilayer conductance due to MelP5 is much higher than the decrease due to either melittin or by the potent equilibrium pore-forming peptide, alamethicin. This result prompted us to develop two novel assays for macromolecule leakage from vesicles and use them to characterize MelP5, melittin and alamethicin. Under conditions where osmotic lysis does not occur, MelP5 allows the passage of macromolecules across vesicle membranes at peptide to lipid ratios as low as 1:100. Neither the melittin nor alamethicin release macromolecules significantly under these conditions. Therefore, the macromolecule-sized, equilibrium pores forms by MelP5 are unique, and MelP5 appears to belong to a novel functional class of peptide with many potential biotechnological applications.