Biophysical characterization of the insertion of two potent antimicrobial peptides-Pin2 and its variant Pin2[GVG] in biological model membranes
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The aim of this study was to investigate the factors that govern the activity and selectivity of two potent antimicrobial peptides (AMPs) using lipid membrane models of bacterial, erythrocyte and fungal cells. These models were used in calcein liposome leakage experiments to explore peptide efficiency. The AMPs (Pin2 and its variant Pin2[GVG]) showed highest affinity towards the bacterial models in the nanomolar range, followed by the erythrocyte and fungal systems. The presence of sterols modulated the variant's selectivity, while the wild type was unaffected. Liposome leakage experiments with Fluorescein Isothiocyanate-dextran (FITC)-dextran conjugates indicated that pore size depended on peptide concentration. Dynamic Light Scattering revealed peptide aggregation in aqueous solution, and that aggregate size was related to activity. The interacting peptides did not alter liposome size, suggesting pore forming activity rather than detergent activity. Atomic Force Microscopy showed differential membrane absorption, being greater in the bacterial model compared to the mammalian model, and pore-like defects were observed. Electrophysiological assays with the Tip-Dip Patch Clamp method provided evidence of changes in the electrical resistance of the membrane. Membrane potential experiments showed that liposomes were also depolarized in the presence of the peptides. Both peptides increased the Laurdan Generalized Polarization of the bacterial model indicating increased viscosity, on the contrary, no effect was observed with the erythrocyte and the fungal models. Peptide membrane insertion and pore formation was corroborated with Langmuir Pressure-Area isotherms and Brewster Angle Microscopy. Finally, molecular dynamics simulations were used to get an insight into the molecular mechanism of action. © 2019 Elsevier B.V.
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Antimicrobial peptides; Biophysical characterization; Lipid membrane models; Lipid-protein interaction; Pin2; Pin2[GVG] adipophilin; calcein; fluorescein isothiocyanate dextran; liposome; polypeptide antibiotic agent; antimicrobial cationic peptide; liposome; sterol; aqueous solution; Article; atomic force microscopy; blood viscosity; Brewster angle microscopy; calculation; controlled study; erythrocyte; fungal cell; genetic variability; intermethod comparison; liposome membrane; microbial activity; molecular dynamics; nonhuman; patch clamp technique; photon correlation spectroscopy; pore size; priority journal; protein analysis; protein interaction; wild type; animal; bacterium; cell membrane; chemistry; drug effect; erythrocyte membrane; fungus; membrane fluidity; membrane potential; viscosity; Animals; Antimicrobial Cationic Peptides; Bacteria; Cell Membrane; Erythrocyte Membrane; Fungi; Membrane Fluidity; Membrane Potentials; Sterols; Unilamellar Liposomes; Viscosity
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