Fine‐tuning the Selectivity of Pore‐forming Peptides from Loss‐of‐function High Throughput Screening

Abstract

While screening a combinatorial peptide library of pore‐forming peptides for gain‐of‐function, we also screened for loss‐of‐function in parallel. We used melittin, a 26 amino acid long lytic peptide from honey bee venom, as a template. For the loss‐of‐function screen we assayed library members for failure to cause leakage of entrapped contents from lipid vesicles at a peptide to lipid ratio (P:L) of 1:20, ten‐fold higher than the concentration at which melittin efficiently permeabilizes. Surprisingly, about one third of the library members are inactive under these conditions. In the selected loss‐of‐function peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that a single residue change from a leucine to a glycine at position 16 completely prevents lytic activity. Unlike the potently lytic melittin, the loss‐of‐function peptides, including the single site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Circular dichroism spectra reveal the loss‐of‐function peptides to shift in the binding‐folding equilibrium away from the bound, a‐helical state towards the unbound, random coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, a‐helical secondary structure and potent activity of the “negative” peptides. The single site variant had potent, melittin‐like antimicrobial activity against Gram‐positive Staph aureus and Gram‐negative E. Coli, with minimum sterilization concentration (MIC) of 10 μM and 7.5 μM, respectively. These results show that conformational fine‐tuning of pore‐forming peptides is a powerful way to modulate potent cytotoxic peptides toward selective antimicrobial activity.