Synergistic Effect of Phenylalanine Residues Governs the Mechanism of Membrane Disrupting Action of Aurein 1.2

Aurein1.2 is a broad-spectrum antimicrobial peptide secreted by the Australian tree frog <i>Litoria aurea</i>. There are indications that the mechanism of action of aurein1.2 is driven primarily by the two phenylalanine (Phe) residues at the 3 and 13 positions. This study explores the role of these residues by comparing the activity of the wild-type peptide to two alanine-substituted derivatives (ALA3 and ALA13) using fluorescence correlation spectroscopy. It was found that wild-type aurein1.2 binds to all membrane types and is only attenuated by cholesterol and membrane saturation. However, dye leakage experiments demonstrated that aurein1.2 selectively disrupts bacterial-mimetic membranes at 7 μM concentration, while disruption is inhibited by cholesterol in mammalian-mimetic membranes. ALA3 became a nonselective membrane disruptor, while ALA13 had very minimal activity. Our results demonstrate that the N-terminal Phe residue drives membrane binding and overall peptide activity, while the C-terminal Phe residue controls membrane selectivity and specificity in a synergistic manner.