De Novo Hydrocarbon-Stapling Design of Single-Turn α-Helical Antimicrobial Peptides

Abstract

Most existing antimicrobial peptides (AMPs) are α-helical and cationic that exhibit typical amphipathic feature to facilitate efficient interaction with bacterial outer membranes. However, short α-helix is unstable in water, and thus naturally occurring α-helical AMPs are generally long and structurally complex. Here, we attempt to perform de novo design of very simple AMPs with only a single-turn α-helix by using hydrocarbon-stapling technique, which can effectively constrain peptide conformation into helical form. The designed AMPs are heptapeptides that have an additional residue at each end of pentapeptides, the theoretical minimum to define an α-turn. Net charge, amphipathicity, sequence pattern and amino acid composition are systematically considered and examined based on the helical wheel of standard α-helical heptapeptide, which derive a series of potential one-turn unstapled AMP candidates with strong hydrophobic moment and a good balance between cationicity and hydrophobicity. Structural analysis suggests that, however, these designed AMPs cannot spontaneously fold into α-helical conformation, with helicity < 50%. An all-hydrocarbon bridge is stapled across the 2nd and 6th residues of several selected heptapeptides to arbitrarily force their α-helical propensity. Circular dichroism spectroscopy demonstrates that the stapling can largely enhance the helical content of these heptapeptides. Molecular dynamics simulation reveals that, as compared to unstapled peptides, the stapled peptides can more efficiently penetrate into the membrane surface of a lipid bilayer model and swiftly move across the hydrophilic surface layer. Susceptibility test reveals that the stapling can considerably improve the antibacterial potency of designed peptides against both the antibiotic-sensitive and methicillin-resistant S. aureus; permeabilization assay confirms that the stapled peptides generally have a higher permeability on E. coil outer membrane than their unstapled counterparts.