Study of the Interaction of a Novel Semi-Synthetic Peptide with Model Lipid Membranes

Abstract

Most linear peptides directly interact with membranes, but the mechanisms of interaction are far from being completely understood. Here, we present an investigation of the membrane interactions of a designed peptide containing a non-natural, synthetic amino acid. We selected a nonapeptide that is reported to interact with phospholipid membranes, ALYLAIRKR, abbreviated as ALY. We designed a modified peptide (azoALY) by substituting the tyrosine residue of ALY with an antimicrobial azobenzene-bearing amino acid. Both of the peptides were examined for their ability to interact with model membranes, assessing the penetration of phospholipid monolayers, and leakage across the bilayer of large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs). The latter was performed in a microfluidic device in order to study the kinetics of leakage of entrapped calcein from the vesicles at the single vesicle level. Both types of vesicles were prepared from a 9:1 (mol/mol) mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol). Calcein leakage from the vesicles was more pronounced at a low concentration in the case of azoALY than for ALY. Increased vesicle membrane disturbance in the presence of azoALY was also evident from an enzymatic assay with LUVs and entrapped horseradish peroxidase. Molecular dynamics simulations of ALY and azoALY in an anionic POPC/POPG model bilayer showed that ALY peptide only interacts with the lipid head groups. In contrast, azoALY penetrates the hydrophobic core of the bilayers causing a stronger membrane perturbation as compared to ALY, in qualitative agreement with the experimental results from the leakage assays.

Enhancing proline-rich antimicrobial peptide action by homodimerization: influence of bifunctional linker

“…S7 † ), we further elucidated the atomic-level details of a lead PrAMP, octofluorobiphenyl dimer-NHNH 2 7, adsorbed on and permeating through a mixed phospholipid membrane bilayer via molecular dynamics simulations. Although phosphatidylethanolamine (PE), rather than neutral phosphatidylcholine (PC), is more commonly found in bacterial membranes, palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG) mixtures are frequently used as proxies for bacterial membranes in experimental studies and molecular dynamics (MD) simulations of protein- 44 and peptide 45 -lipid interactions, as the use of PC confers the advantage of producing a membrane with less curvature stress 46 compared to PE. For each bilayer adsorption simulation, a pre-equilibrated mixed bilayer composed of POPC and POPG in a 7 : 3 ratio serves as a model of Gram negative inner membranes.…”

Mechanistic Understanding from Molecular Dynamics in Pharmaceutical Research 2: Lipid Membrane in Drug Design

“…In a simulation study carried out by Koivuniemi et al, MD simulations of these derivatives interacting with both prokaryotic and eukaryotic model membranes found evidence of important differences in their behavior in the two membrane types [843]. Additionally, antimicrobial peptides can have their desired properties enhanced through the introduction of non-natural amino acids, e.g., the azoALY peptide derived from ALY (full sequence: ALYLAIRKR) by functionalization of the sole tyrosine residue present with an azobenzene group [844]. Both MD simulations and experimental studies have found evidence that this modification results in a peptide with an enhanced interaction with lipid bilayers.…”

Azobenzene as Antimicrobial Molecules

“…Lipids modified with azobenzene have received particular attention in recent years because photolipids, lipophilic molecules, can be rapidly converted from a linear to a folded form by exploiting the isomerization of the -N=N- double bond. Some authors have also used azobenzene molecules to synthesize semi-synthetic peptides, by evaluating their ability to penetrate through membranes and their behavior through the phospholipid bilayer of GUVs (Giant unilamellar vesicles) and LUVs (large unilamellar vesicles) [ 75 ]. Similarly, azobenzene molecules can be included in polymer matrices, or even be used as pendants covalently linked to polymer chains to obtain materials with various properties, such as antimicrobial.…”