Diverse nanostructures and antimicrobial activity of lipopeptides bearing lysine-rich tripeptide sequences

Abstract

The self-assembly and conformation in aqueous solution and bioactivity of three lipopeptides bearing lysine-rich tripeptide sequences are compared for C₁₆-KFK, C₁₆-KWK, and C₁₆-KYK, where C₁₆ denotes an N-terminal hexadecyl (palmitoyl) chain. The central aromatic residue has a significant effect on the self-assembled nanostructures, since C₁₆-KFK forms nanotubes, whereas the other two lipopeptides form nanotapes. The nanotubes and nanotapes are built from lipopeptide bilayers, as confirmed by small-angle X-ray scattering. Circular dichroism (CD) spectroscopy and thioflavin T dye fluorescence show the presence of β-sheet structures, and the latter technique was used to determine critical aggregation concentrations (CACs). Fibre X-ray diffraction for C₁₆-KFK shows a well-defined helical diffraction pattern arising from the helically wrapped bilayers in the nanotube walls. The lipopeptides act as surfactants, as confirmed by surface tension measurements (also used to determine CAC values). All three lipopeptides show minimal cytotoxicity to human fibroblasts but also, unexpectedly, low activity against Gram-negative and Gram-positive bacteria, in contrast to previously studied analogues (with switch of two residues) C₁₆-WKK and C₁₆-YKK that show significant antimicrobial action with low minimum inhibitory concentration (MIC) values [A. Adak et al., ACS Appl. Bio Mater., 2024, 7, 5553–5565]. Also in contrast to these molecules (which show a transition from micelles to fibrils upon increasing the pH), C₁₆-KFK, C₁₆-KWK, and C₁₆-KYK form extended β-sheet structures over the whole pH range examined (pH 2–8). These observations point to the remarkable sensitivity to the tripeptide pattern of lipopeptide self-assembly and antibacterial activity. Whereas the C₁₆-XKK (X = W or Y) lipopeptides form cylindrical fibrils, the C₁₆-KXK analogues form bilayer nanotapes. The former show significant toxicity to bacteria in contrast to the latter, which we propose is due to the effect of the lipopeptide assembly curvature on induced bacterial membrane deformation.