Translocation of a Channel-Forming Antimicrobial Peptide, Magainin 2, across Lipid Bilayers by Forming a Pore

Matsuzaki, Katsumi; Murase, Osamu; Fujii, Nobutaka; Miyajima, Koichiro

doi:10.1021/bi00019a033

Cited by 311 publications

(336 citation statements)

References 40 publications

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“…Unfortunately, our lysoMC-RET method cannot be used for the determination of melittin topology in POPC vesicles because it induces rapid transbilayer equilibration of lysoMC at concentrations as low as 0.1 mol % ( Figure 6). This may, of course, be an indirect indication that melittin at least transiently crosses the bilayer, as implied by other experiments (12,35,(52)(53)(54)(55). In this light, it is interesting that 0.5 mol % melittin does not induce lysoMC flip-flop nor does it transit the bilayer to the inner leaflet in POPG vesicles (T ∼ 1), consistent with the observation that melittin does not form sizable pores in POPG vesicles (Ladokhin, A. S., and White, S. H., unpublished observation).…”

Section: Discussionsupporting

confidence: 54%

Determining the Membrane Topology of Peptides by Fluorescence Quenching

Wimley

White

1999

Biochemistry

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Determination of the topology of peptides in membranes is important for characterizing and understanding the interactions of peptides with membranes. We describe a method that uses fluorescence quenching arising from resonance energy transfer ("FRET") for determining the topology of the tryptophan residues of peptides partitioned into phospholipid bilayer vesicles. This is accomplished through the use of a novel lyso-phospholipid quencher (lysoMC), N-(7-hydroxyl-4-methylcoumarin-3-acetyl)-1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine. The design principle was to anchor the methylcoumarin quencher in the membrane interface by attaching it to the headgroup of lyso-phosphoethanolamine. We show that lysoMC can be incorporated readily into large unilamellar phospholipid vesicles to yield either symmetrically (both leaflets) or asymmetrically (outer leaflet only) labeled bilayers. LysoMC quenches the fluorescence of membrane-bound tryptophan by the Förster mechanism with an apparent R 0 that is comparable to the thickness of the hydrocarbon core of a lipid bilayer (∼25 Å). Consequently, the methylcoumarin acceptor predominantly quenches tryptophans that reside in the same monolayer as the probe. The topology of a peptide's tryptophan in membranes can be determined by comparing the quenching in symmetric and asymmetric lysoMC-labeled vesicles. Because it is essential to know that asymmetrically incorporated lysoMC remains so under all conditions, we also developed a second type of FRET experiment for assessing the rate of transbilayer diffusion (flip-flop) of lysoMC. Except in the presence of poreforming peptides, there was no measurable flip-flop of lysoMC, indicating that asymmetric distributions of quencher are stable. We used these methods to show that N-acetyl-tryptophan-octylamide and tryptophanoctylester rapidly equilibrate across phosphatidylcholine (POPC) and phosphatidylglycerol (POPG) bilayers, while four amphipathic model peptides remain exclusively on the outer monolayer. The topology of the amphipathic peptide melittin bound to POPC could not be determined because it induced rapid flip-flop of lysoMC. Interestingly, melittin did not induce lysoMC flip-flop in POPG vesicles and was found to remain stably on the external monolayer.Peptide model systems are increasingly being used for the elucidation of the principles of the insertion, folding, and structure of proteins and peptides in membranes (reviewed in 1-4). A critical issue is the topology of peptides incorporated into lipid bilayers: does a particular model peptide equilibrate freely across the bilayer, form a stable transmembrane structure, or remain only on one surface? The answer to this question clarifies the physicochemical state of a peptide in membranes and thus bears upon the biological activity of peptide model systems. The question is infrequently addressed, however, because there are only a few reliable methods for answering it. The methods fall into three classes. The most widely utilized methods include oriented diffra...

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Section: Discussionsupporting

confidence: 54%

Determining the Membrane Topology of Peptides by Fluorescence Quenching

Wimley

White

1999

Biochemistry

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“…The translocation occurs upon the disintegration of a pore (24). The translocation efficiency is known to be enhanced by increasing the peptide's positive charge, which destabilizes the intermediate pore (29).…”

Section: Resultsmentioning

confidence: 99%

“…Upon pore disintegration, a fraction of the peptide molecules translocates across the membrane (24). This is reflected by a cooperativity in peptide binding, membrane permeabilization (28), and cytotoxicity (22).…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, short Arg-rich cationic peptides have recently been shown to enter mammalian cells by an energy (ATP)-independent, non-endocytotic pathway and have been utilized as vectors for delivering membrane-impermeable drugs, oligonucleotides, and proteins into cells (23). Model membrane studies revealed that the membrane-acting magainin peptide also translocates across lipid bilayers (24,25), although less effectively than buforin 2 (18). Therefore, cationic antimicrobial peptides, including those of the membrane-permeabilizing class, can penetrate human cells.…”

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confidence: 99%

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Translocation of Analogues of the Antimicrobial Peptides Magainin and Buforin across Human Cell Membranes

Takeshima

Chikushi

Lee

et al. 2003

Journal of Biological Chemistry

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185

151

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“…In the toroidal-pore model (Matsuzaki et al, 1995, peptides first reorient with membrane lipid. Similar to the barrel-stave model, peptides insert perpendicularly to the membrane to form a pore, while the hydrophilic regions of the peptides keep the association with lipid head groups and the hydrophobic peptide regions associate with the hydrophobic core of the membrane lipids.…”

Section: Mechanism Of Actionmentioning

confidence: 99%

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

2010

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Antimicrobial peptides (AMPs), with their extraordinary properties, such as broad-spectrum activity, rapid action and difficult development of resistance, have become promising molecules as new antibiotics. Despite their various mechanisms of action, the interaction of AMPs with the bacterial cell membrane is the key step for their mode of action. Moreover, it is generally accepted that the membrane is the primary target of most AMPs, and the interaction between AMPs and eukaryotic cell membranes (causing toxicity to host cells) limits their clinical application. Therefore, researchers are engaged in reforming or de novo designing AMPs as a 'singleedged sword' that contains high antimicrobial activity yet low cytotoxicity against eukaryotic cells. To improve the antimicrobial activity of AMPs, the relationship between the structure and function of AMPs has been rigorously pursued. In this review, we focus on the current knowledge of α-helical cationic antimicrobial peptides, one of the most common types of AMPs in nature.

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Translocation of a Channel-Forming Antimicrobial Peptide, Magainin 2, across Lipid Bilayers by Forming a Pore

Cited by 311 publications

References 40 publications

Determining the Membrane Topology of Peptides by Fluorescence Quenching

Determining the Membrane Topology of Peptides by Fluorescence Quenching

Translocation of Analogues of the Antimicrobial Peptides Magainin and Buforin across Human Cell Membranes

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

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