Beyond Structural Pores: Transient Permeabilization of Lipid Membranes by Antimicrobial Peptides

Antimicrobial peptides (AMPs) are highly effective broad-spectrum antibiotics, found as components of the immune system in almost all forms of life. It is well established that their primary target is the cell membrane where they are hypothesized to permeabilize the bilayer. However, it is a long-standing question whether stable transmembrane and structural pores are necessary, or if the peptides permeabilize the membrane through transient channels. Here this question is addressed with simultaneous characterization of the membrane structure, peptide partitioning, and ion transport in real time, using timeresolved small-angle X-ray and neutron scattering (TR-SAXS and TR-SANS). These experiments reveal that even unstructured and non-penetrating AMPs can rapidly permeabilize the membrane, with complete ion equilibration occurring in only a few tens of milliseconds. Fully-atomistic molecular dynamics (MD) simulations show that the peripherally bound peptides disrupt the lipid packing and cause an increased flip-flop rate which results in transient aqueous pores. A diffusion model based on these transient pores is developed and fitted to the experimentally measured kinetics, showing excellent correlation between experiment and theory. This label-free approach, with millisecond time resolution, demonstrates that structural pores are not required and represents a significant step toward illuminating the much-debated molecular mechanisms of AMPs.