The mechanism of action of antimicrobial peptides is, to our knowledge, still poorly understood. To probe the biophysical characteristics that confer activity, we present here a molecular-dynamics and biophysical study of a cyclic antimicrobial peptide and its inactive linear analog. In the simulations, the cyclic peptide caused large perturbations in the bilayer and cooperatively opened a disordered toroidal pore, 1-2 nm in diameter. Electrophysiology measurements confirm discrete poration events of comparable size. We also show that lysine residues aligning parallel to each other in the cyclic but not linear peptide are crucial for function. By employing dual-color fluorescence burst analysis, we show that both peptides are able to fuse/aggregate liposomes but only the cyclic peptide is able to porate them. The results provide detailed insight on the molecular basis of activity of cyclic antimicrobial peptides.

We report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a β-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides.

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis.