Conformational Changes of Anoplin, W-MreB1–9, and (KFF)3K Peptides near the Membranes

Abstract

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB1–9, and one cell-penetrating peptide, (KFF)3K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB1–9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB1–9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.

Stapled Anoplin as an Antibacterial Agent

“…CD was used to determine the secondary structure of the peptides in the buffer solution ( Figure 1C ) and the environments mimicking membranes ( Figure 2 ). As shown earlier ( Konno et al, 2001 ; Wojciechowska et al, 2020 ), in the phosphate buffer, anoplin adopted a random coil ( Figure 1C ; Table 1 ). As expected, the introduction of staples into the anoplin sequence induced the α-helical structure ( Figure 1C red and green lines).…”

“…Many research groups investigated multiple modifications of anoplin to improve its pharmacological activity ( Wu et al, 2020 ). Our studies showed low antibacterial activity of this peptide against Escherichia coli strains and confirmed that anoplin adopts an α-helical conformation only near the lipid environments ( Wojciechowska et al, 2020 ). Therefore, we hypothesized that stabilizing the α-helix of anoplin would improve its antibacterial activity.…”

“…DPC micelles and SUVs composed of POPE and POPG lipids are neutral, so they resemble eukaryotic membrane mimics. The POPC:POPG or POPC:POPE mixtures were used in a molar ratio of 3:1 ( Russell et al, 2012 ; Wojciechowska et al, 2020 ). We also investigated the change of peptide secondary structure in the presence of LPS isolated from the E. coli O111:B4 strain ( Avitabile et al, 2015 ).…”

“…CD spectra were recorded in aqueous buffer solution (10 mM phosphate buffer, pH 7.0), in the presence of 5 mM SDS, 2 mM dodecylphosphocholine (DPC), 50 μM LPS micelles, and 1 mM POPC:POPG (3:1) or 1 mM POPC:POPE (3,1) phospholipids, as described in Wojciechowska et al (2020 ). In all CD experiments, the peptide concentrations were 120 μM.…”

“…Our interests focused on the peptides that primarily take on an active structure only near the lipid environment and close to the bacterial cells ( Wojciechowska et al, 2020 ). A promising approach to improve the pharmacological properties of AMP may be stabilizing their active conformation.…”