magainin-2-peptide--xenopus and fluorexon

It is thought that magainin 2, an antimicrobial peptide, acts by binding to lipid membranes. Recent studies using a suspension of large unilamellar vesicles (LUVs) indicate that magainin 2 causes gradual leakage from LUVs containing negatively charged lipids. However, the details of the characteristics of the membrane permeability and the mechanism of pore formation remain unclear. In this report, we investigated the interaction of magainin 2 with single giant unilamellar vesicles (GUVs) composed of a dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol mixture (50% DOPG/50% DOPC GUVs) containing the fluorescent dye, calcein, by phase contrast, fluorescence microscopy using the single GUV method. Low concentrations (3-10 microM) of magainin 2 caused the rapid leakage of calcein from single GUVs but did not disrupt the liposomes or change the membrane structure, showing directly that magainin 2 forms membrane pores through which calcein leaked. The rapid leakage of calcein from a GUV started stochastically, and once it began, the complete leakage occurred rapidly (6-60 s). The fraction of completely leaked GUV, P(L), increased with time and also with an increase in magainin 2 concentration. Shape changes in these GUVs occurred prior to the pore formation and also at lower concentrations of magainin 2, which could not induce the pore formation. Their analysis indicates that binding of magainin 2 to the external monolayer of the GUV increases its membrane area, thereby raising its surface pressure. The addition of lysophosphatidylcholine into the external monolayer of GUVs increased P(L). On the basis of these results, we propose the two-state transition model for the pore formation.

Topics: Antimicrobial Cationic Peptides; Fluoresceins; Liposomes; Lysophosphatidylcholines; Magainins; Permeability; Phosphatidylglycerols; Xenopus Proteins

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Biological Transport; Cattle; Dose-Response Relationship, Drug; Drug Delivery Systems; Filaggrin Proteins; Fluoresceins; Indicators and Reagents; Intermediate Filament Proteins; Lipid Bilayers; Magainins; Skin; Xenopus laevis; Xenopus Proteins

Magainin 2 and tachyplesin I (T-SS) are membrane-permeabilizing antimicrobial peptides discovered from frog skin and horseshoe crab hemolymph, respectively. They are classified into different secondary structural classes, i.e., alpha-helix and cyclic beta-sheet, respectively. We found that F5W-magainin 2 (MG2) and T-SS exhibited marked synergistic effects against Gram-negative and Gram-positive bacteria without enhancing hemolytic activity as a measure of toxicity. Dye release experiments using liposomes suggested that the selective synergism is mainly due to anionic phospholipid-specific synergism in membrane permeabilization. Furthermore, the cyclic structure of T-SS was found to be necessary for synergism because a linear analogue of T-SS did not show good synergism with MG2. These novel observations suggested the possibility of the development of cocktail therapeutic regimens using combinations of antimicrobial peptides.

Topics: Adult; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Binding Sites; Cell Membrane; Circular Dichroism; DNA-Binding Proteins; Drug Synergism; Erythrocytes; Female; Fluoresceins; Gram-Negative Bacteria; Gram-Positive Bacteria; Hemolysis; Horseshoe Crabs; Humans; Magainins; Peptides, Cyclic; Phospholipids; Protein Conformation; Skin; Xenopus laevis; Xenopus Proteins

The membrane-disrupting properties of cecropin A (1-8)-magainin 2 (1-12) hybrid peptide, which has higher antitumor with less hemolytic activities than cecropin A (1-8)-melittin (1-12), and its analogues were assessed by measuring the induced release of vesicle-entrapped fluorescence probes. A model membrane was made of zwitterionic phospholipid (phosphatidylcholine) or the mixture of negatively and zwitterionic phospholipids (phosphatidylcholine and phosphatidylserine). The extent of leakage of the aqueous content of the phospholipid vesicles was found to have remarkable discrepancies according to the amphipathic nature of each analogue peptide. The entrapped high molecular weight solute (fluorescein-labeled immunoglobulin G, 55 kDa) also was released by the analogue which had the largest hydrophobic region and the highest amphipathic score among peptides tested. As the result of the determination of the relationships between the membrane-disrupting properties and the hydrophobicity values of peptides, it was found that the membrane-disrupting activity increased according to increasing the hydrophobicity of the peptide. The tryptophan fluorescence emission spectra and CD spectra showed that on interaction with the phospholipid vesicle, the peptide acquired the ordered structure and alpha-helical conformation by moving a tryptophan residue into the nonpolar environment of the phospholipid vesicle. These results suggest that the breakdown of the lipid bilayer was mediated by the alpha-helical amphipathic structure of the peptide interacting with the lipid bilayers as well as the by the hydrophobicity of the peptide.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Circular Dichroism; Fluoresceins; Immunoglobulin G; Liposomes; Magainins; Molecular Sequence Data; Peptide Fragments; Peptides; Phospholipids; Protein Structure, Secondary; Recombinant Fusion Proteins; Spectrometry, Fluorescence; Xenopus Proteins

The magainins are antibacterial peptides from the skin of Xenopus laevis. They show a broad range of activity against prokaryotic cells but lyse eukaryotic cells poorly. To elucidate the influence of peptide hydrophobicity on membrane activity and selectivity, we designed and synthesized analogs of magainin 2 amide with slightly varying hydrophobicities but retained hydrophobic moment, peptide charge, and angle subtended by the hydrophilic helix region. Circular dichroism investigations of the peptides revealed that all peptides investigated adopt an alpha-helical conformation when bound to phospholipid vesicles. Dye-releasing experiments from vesicles of phosphatidylglycerol (PG) showed that the membrane-permeabilizing activity of the analogs is not influenced by peptide hydrophobicity. In contrast, the permeability-enhancing activity on vesicles bearing high amounts of phosphatidylcholine (PC) increases drastically with enhanced peptide hydrophobicity, resulting in a reduced selectivity of more hydrophobic analogs for negatively charged membranes. Likewise, the peptide affinity to PC-rich membranes increases in the order of hydrophobicity. Correlation of peptide binding and membrane permeabilization of PC/PG (3:1) vesicles revealed that the observed differences in peptide activity on membranes of low negative surface charge are mainly caused by the different binding affinities. The antibacterial and hemolytic activity of the peptides increases with enhanced hydrophobicity. A strong correlation was found between the hemolytic effect and the bilayer-permeabilizing activity against PC-rich vesicles. Whereas the antibacterial specificity of the more hydrophobic analogs is retained for Escherichia coli, the specificity for Pseudomonas aeruginosa decreases with increasing hydrophobicity.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Circular Dichroism; Fluoresceins; Gram-Negative Bacteria; Hemolysis; Humans; Lipid Bilayers; Magainins; Microbial Sensitivity Tests; Peptides; Permeability; Phosphatidylcholines; Phosphatidylglycerols; Protein Binding; Protein Structure, Secondary; Structure-Activity Relationship; Xenopus Proteins

The effect of an antimicrobial peptide, magainin 2, on the flip-flop rates of phospholipids was investigated by use of fluorescent lipids, i.e., anionic N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (NBD-PE), 1-oleoyl-2-[12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)- dodecanoyl]-L-alpha-phosphatidic acid (C12-NBD-PA), 1-oleoyl-2-[12- ((7-nitrobenz-2-oxa-1,3-diazol-4-yl)- amino)dodecanoyl]-L-alpha-phosphatidyl-L-serine (C12-NBD-PS), and zwitterionic 1-palmitoyl-2-[6-((7- nitrobenz-2-oxa-1,3-diazol-4-yl)amino)caproyl]-L-alpha-phosphatidy lcholine (C6-NBD-PC). Their intrinsic flip-flop half-lives at 30 degrees C in the absence of the peptide were 1.1 h, ca. 7 h, ca. 8 days, and > 2 days, respectively. The peptide accelerated the flip-flop half-lives of the fluorescent lipids to an order of minutes. Furthermore, the flip-flop was coupled with the membrane permeabilization and the peptide translocation [Matsuzaki, K., Murase, O., Fujii, N., & Miyajima, K. (1995) Biochemistry 34, 6521-6526], suggesting pore-mediated flip-flop. The flip-flop rate was independent of the initial labeling conditions (outer leaflet label or inner leaflet label). From these results, a model was proposed, in which the lipids translocate across the membrane by lateral diffusion along the wall of the pores composed of the peptides and the lipids. A simple theoretical calculation could explain the coupling of the flip-flop with the permeabilization.

Topics: Amino Acid Sequence; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Diffusion; Dithionite; Fluoresceins; Fluorescent Dyes; Kinetics; Lipid Bilayers; Liposomes; Magainins; Mathematics; Molecular Sequence Data; Mutation; Peptides; Permeability; Phosphatidylethanolamines; Phospholipids; Skin; Xenopus laevis; Xenopus Proteins

The kinetics of the pore formation by magainin 2, an antimicrobial peptide from Xenopus laevis, in lipid vesicles was investigated. The pore formation was estimated by the efflux of a fluorescent dye, calcein, from large unilamellar vesicles composed of egg yolk phosphatidylglycerol. The time courses of the dye release were well-described by a novel model in which the peptide molecules translocate from the outer to the inner monolayer by forming a pore. The concentration dependence of the leakage rate suggested that the pore consists of pentameric magainin. The obtained kinetic parameters estimate that, at a lipid-to-peptide molar ratio of 117, 9 pores with a lifetime of 40 microseconds open per second per vesicle in the initial phase. The apparent deactivation of the pore with increasing time can be ascribed to the reduced peptide density in the outer leaflet due to the translocation. Incorporation of phosphatidylcholine destabilized the pore, indicating the importance of anionic lipids in the stable pore formation.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Anti-Infective Agents; Antimicrobial Cationic Peptides; Antineoplastic Agents; Cell Membrane Permeability; Dithionite; Fluoresceins; Kinetics; Lipid Bilayers; Magainins; Molecular Sequence Data; Phospholipids; Xenopus laevis; Xenopus Proteins