Similarities and differences for membranotropic action of three unnatural antimicrobial peptides

Previously, we described the design and synthesis of three nine‐residue AMPs, P9Nal(SS), P9Trp(SS), and P9Nal(SR), showing high stability in serum and broad spectrum antimicrobial activity. The peptides P9Trp(SS) and P9Nal(SR) differ from P9Nal(SS) for the replacement of the two 2Nal residues with Trp residues and for the replacement of the two Cys (StBu) with Cys (tBu) residues, respectively. These changes led to peptides with a lower hydrophobicity respect to the P9Nal(SS). Interestingly, the three peptides have very similar activity against Gram‐negative bacteria. Instead, they exhibit a significant difference towards Gram‐positive bacteria, being P9Nal(SS) the most active. In order to evaluate the impact of amino acids substitution on membranotropic activity and rationalize the observed effects in vivo, here, we report the detailed biophysical characterization of the interaction between P9Nal(SR) and P9Trp(SS) and liposomes by combining differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The comparison with the results for the previously characterized P9Nal(SS) peptide reveals similarities and differences on the interaction process and perturbation activities. It was found that the three peptides can penetrate at different extent inside the bilayer upon changing their conformation and inducing lipid domains formation, revealing that the formation of lipid domains is fundamental for the activity against Gram‐negative bacteria. On the contrary, the dissimilar activity against Gram‐positive bacteria well correlate with the different affinity of peptides for the lipoteichoic acid, a component selectively present in the cell wall of Gram‐positive bacteria. Three analog antimicrobial peptides containing unnatural amino acids, through a lipid segregation process, are able to penetrate at different extent inside bacterial model membranes.