Melittin induced voltage-dependent conductance in DOPC lipid bilayers

Pawlak, Michael; Stankowski, Stefan; Schwarz, Gerhard

doi:10.1016/0005-2736(91)90339-a

Cited by 63 publications

(63 citation statements)

References 38 publications

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“…Additional data of the channel characteristics of melittin in pure DOPC are taken from the literature (Pawlak, 1991;Pawlak et al, 1991). The membrane activity of melittin as revealed by electrical measurements can be characterized by 3 typical effects, shown in Figure 11.…”

Section: Native Melittinmentioning

confidence: 99%

“…Macroscopic membrane conductance, induced by a statistically significant number of channels, was studied on decanecontaining BLMs (Mueller et al, 1962) with large membrane areas (0.4 mm2). The bilayers were formed from DOPC as described by Pawlak et al (1991). The membranes separated 2 compartments (each of 10 mL of volume) in order to minimize protein adsorption to the walls.…”

Section: Conductance Measurements On Planar Lipid Bilayersmentioning

confidence: 99%

“…1). Melittin itself, a 26-amino acid peptide from bee venom, induces channel activity in lipid bilayers that is thought to be dominated by the formation of tetrameric a-helix bundles (Tosteson & Tosteson, 1981;Pawlak et al, 1991). Both the electrical properties of the polypeptide channels and the structural characteristics of melittin have been the subject of intensive research during the last 15 years (for a review, see Dempsey, 1990).…”

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Template‐assembled melittin: Structural and functional characterization of a designed, synthetic channel‐forming protein

et al. 1994

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Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin,_,,-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid temp1at.e were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt a-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A ' , compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 X lo9 M" was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaC1). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltagedependent channet activity OR membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 1 0 0 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation. Abbreviations: 6-CF, 6-carboxyfluorescein; BLM, black lipid membranes; Boc. tert-butyloxycarbonyl; BOP, benzotriazol-I-yl-oxybis(dimethy1amino)phosphonium hexafluorophosphate; DCM, dichloromethane; DIC, diisopropylcarbodiimide; DIPEA, N-diisopropylethylamine; DMF, dimethylformarnide; DMS, dimethylsulfide; DOPC, 1,2-dioleoyl-snglycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycer0-3-phosphoethanolamine; DTPC, 1,2-ditetradecyl-sn-glycero-3-phosphocholine; EDT, ethanedithiole; Fmoc, 9-fluorenylmethyloxycarbonyl; HOBt, 1-hydroxybenzotriazole; L:P, lipid to protein molar ratio; MBHA, methylbenzhydrylamine; Mob, 4-methoxybenzyl; Mtr, 4-methoxy-2,3,6-trimethylbenzenesulfonyl; POPC, I-palmitoyl-2-0leoyl-sn-glycero-3-phosphocholine; RP-HPLC, reverse-phase high-performance liquid chromatography; SUV, LUV, small, large unilamellar vesicle(s); TASP, template-assembled synthetic protein; tBu, tert-butyl ether; TFA, trifluoroacetic acid; TFMSA, trifluoromethanesulfonic acid; T...

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Section: Native Melittinmentioning

confidence: 99%

Section: Conductance Measurements On Planar Lipid Bilayersmentioning

confidence: 99%

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Template‐assembled melittin: Structural and functional characterization of a designed, synthetic channel‐forming protein

et al. 1994

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“…4B). This characteristic conductance is comparable to that of the channels formed by natural cytotoxic peptides like melittin and insect defensins, which fall in the range 100-200 pS (Pawlak et al, 1991;Cociancich et al, 1993).…”

Section: Sequence Confirmation and Conformational Analysismentioning

confidence: 58%

Structural Design and Characterization of a Channel-forming Peptide

Krittanai¹,

Panyim²

2004

BMB Reports

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A 16-residue polypeptide model with the sequence acetyl-YALSLAATLLKEAASL-OH was derived by rational de novo peptide design. The designed sequence consists of amino acid residues with high propensity to adopt an alpha helical conformation, and sequential order was arranged to produce an amphipathic surface. The designed sequence was chemically synthesized using a solid-phase method and the polypeptide was purified by reverse-phase liquid chromatography. Molecular mass analysis by electro-spray ionization mass spectroscopy confirmed the correct designed sequence. Structural characterization by circular dichroism spectroscopy demonstrated that the peptide adopts the expected alpha helical conformation in 50% acetonitrile solution. Liposome binding assay using Small Unilamellar Vesicle (SUV) showed a marked release of entrapped glucose by interaction between the lipid membrane and the tested peptide. The channel-forming activity of the peptide was revealed by a planar lipid bilayer experiment. An analysis of the conducting current at various applied potentials suggested that the peptide forms a cationic ion channel with an intrinsic conductance of 188 pS. These results demonstrate that a simple rational de novo design can be successfully employed to create short peptides with desired structures and functions.

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“…Single-channel conductances of insect defensin are also smaller. For example, the single channel conductance value of melittin (a constituent of bee venom) was about 11OpS in 1.8M KC1 (Pawlak et al, 1991). In contrast, antimicrobial peptides from amphibians give closer values.…”

Section: Discussionmentioning

confidence: 99%

Characterization and Ion Channel Activities of Novel Antibacterial Proteins from the Skin Mucosa of Carp (Cyprinus carpio)

Lemaître

Orange²,

Saglio³

et al. 1996

European Journal of Biochemistry

106

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A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers. A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31 -kDa and 27-kDa proteins. In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave P J P , ratios of 0.6 and 1.0, respectively. These proteins had large potent microbicidal activities (0.018 -0.1 8 pM) against different strains of gramnegative and gram-positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia ; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp. ; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a 19-amino-acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.

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Melittin induced voltage-dependent conductance in DOPC lipid bilayers

Cited by 63 publications

References 38 publications

Template‐assembled melittin: Structural and functional characterization of a designed, synthetic channel‐forming protein

Template‐assembled melittin: Structural and functional characterization of a designed, synthetic channel‐forming protein

Structural Design and Characterization of a Channel-forming Peptide

Characterization and Ion Channel Activities of Novel Antibacterial Proteins from the Skin Mucosa of Carp (Cyprinus carpio)

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