Tryptophan substitutions reveal cooperative membrane transitions and aromatic interactions associated with membrane permeabilisation in CM15 and MSI-78 peptide analogues.

Antimicrobial peptides CM15 (Ac-KWKLFKKIGAVLKVL-NH2) and MSI-78(11-22)-NH₂ (Ac-CKFGKAFVKILKK-NH2) have remarkable abilities to interact with negatively charged lipid bilayers, adopting helical secondary structures while embedded within the membrane, and, with increasing peptide concentration, evolving toward aggregation and membrane permeabilising states. Membrane permeabilisation has been attributed to peptide antibacterial activity, but due to the complexity of the peptide-membrane interaction there are no simple models to define the concentration threshold required for each process. In the present report, we modified CM15 by substituting hydrophobic leucine and isoleucine residues with tryptophan and tyrosine at positions 4, 8, or 12, as a strategy for modulating peptide-membrane and peptide self-interaction with hydrophobic aromatic residues. Evaluation of the peptide's interaction with POPE:POPG (70:30) large unilamellar vesicles (LUVs) by fluorescence emission and anisotropy of tryptophan residues permitted identifying a peptide to lipid ratio (P/L) range consistent with a transition from an interfacial peptide orientation to a deeper inserted configuration or peptide assembly states in the membrane, in agreement with zeta potential measurements displaying a cooperative peptide-membrane association. The amino acid modifications on the CM15 derivatives influenced the peptide interaction with the membrane but had a moderate effect on the peptide concentration required for calcein release or to reach the minimal inhibitory concentration against Staphylococcus aureus or Escherichia coli. An alternative approach assessed was using Ac-Cys-MSI-78(11-22)-NH₂ (Ac-CKFGKAFVKILKK-NH₂) containing an N-terminal Cys residue to enable covalent dimerisation through disulfide bonds, together with substitution of phenylalanine for tryptophan at positions 3 or 7 to promote intramolecular tryptophan-tryptophan interaction between both peptide segments. The dimer with tryptophan in position 3 exhibited more efficient calcein leakage than its simile with tryptophan in position 7, consistent with enhanced tryptophan π-aromatic interactions between peptide segments. Molecular dynamics studies of selected CM15 derivatives and MSI-78(11-22) dimers in a lipid membrane model revealed persistent interactions between lysine residues and negatively charged headgroups, as well as contacts between hydrophobic residues and lipid tails. These simulations also showed sequence-dependent differences in peptide insertion and helical stability, providing a structural framework to interpret the experimentally observed differences in membrane permeabilisation trends. Oxidation product patterns of peptides in LUVs induced by water-soluble oxygenated free radicals favoured monomer di-tyrosine dimerisation, supporting the formation of peptide assemblies associated to the membrane and localized lipidic defects that may enhance calcein release and contribute with the observed antimicrobial activity.