Structural analysis of four cyclic antimicrobial hexapeptides in aqueous solution and in micelles, towards membrane-mimicking nanodiscs

Cationic, antimicrobial peptides (AMPs) are abundantly present in nature as host-defensive peptides, forming the backbone of the natural defence of many organisms. Their primary interaction with bacterial membranes is thought to be a binding to the outer layer, followed by a disruption of the lipid membrane. Investigating the mode-of-action of AMPs is an ever-growing field, where a plethora of different biophysical experiments are used to investigate this. Here, we explore the experimental limitation in NMR spectroscopy as we migrate towards increasingly realistic but larger membrane model systems with the aim to scout which data we could realistically extract from AMPs interacting with self-aligning nanodiscs. A set of cyclic hexapeptides were used as model compounds, in water, DMSO, and SDS micelles. The quality of isotropic and anisotropic NMR parameters decreased as the result of faster relaxation due to size and dynamics, as well as the need for water suppression and buffer salts. Chemical shifts, scalar couplings, NOE derived distances, and simulated annealing suggested that the backbone conformation of both peptides remained mostly rigid, forming two β-turns, while the sidechains remain flexible in all environments. The difference between Lys or Arg did not change the backbone conformation, even though the two peptides have different MIC concentrations, indicating that amino acid composition is more important than conformation for antimicrobial activity. Theoretical structures in an explicit lipid bilayer were simulated showing similar structures. Pilot experiments on SMA-QA nanodiscs indicated that they are viable as membrane model for investigating the interaction between AMPs and lipid membranes. It was shown that signal can be obtained from bound peptide, but further investigations with isotopically labelled peptides are needed to extract relevant structural data under conditions where the nanodisc is not over-saturated by peptides.