Physical Studies of Phospholipid/Alamethicin Interactions

Chapman, D.; Cherry, Richard J.; Finer, E.G.; Häuser, H.; Phillips, Michael C.; Shipley, G. Graham; McMullen, A.I.

doi:10.1038/224692a0

Cited by 38 publications

(15 citation statements)

References 7 publications

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“…A parallel series of digests were set up in buffers that contained 10% (v/v) ethanol in the hope that this might disaggregate the substrate (Chapman et al 1969). Controls containing 0.5mg of the B-chain of oxidized insulin (a gift from Dr F. Sanger) were digested in parallel to confirm that the enzymes were active under the experimental conditions, and further controls in which substrate was omitted allowed for any autolysis of the enzymes themselves.…”

Section: Methodsmentioning

confidence: 99%

“…Each stained yellow with cadmium-ninhydrin reagent and blue with isatin, indicating N-terminal proline, and each appeared to be a single component on further electrophoresis at pH 3.5. Reaction with chlorinetolidine reagent revealed an additional intense band at the origin, which is presumably unhydrolysed aggregated alamethicin (Chapman et al 1969). Amino acid analyses (Table 1) showed that peptide 1 had a composition identical with that of alamethicin itself, and peptides 2 and 3 had compositions that together account for the 18 amino acid residues.…”

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confidence: 99%

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The primary structure of alamethicin

Payne

Jakes

Hartley

1970

Biochemical Journal

129

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Alamethicin, an antibiotic that can transport cations and induce action potentials in synthetic membranes, is shown to be a cyclic peptide with 18 residues including 7-alpha-aminoisobutyric acid residues, two glutamine residues and one free carboxyl group. The composition indicates microheterogeneity. Alamethicin itself and many peptides derived from it are immune to enzymic digestion, but specific partial acid cleavages have allowed determination of the complete sequence. Diborane reduction has shown that the alpha-carboxyl group of glutamine-18 is free, but the ring is formed by a peptide bond between the imino group of proline-1 and the gamma-carboxyl group of glutamic acid-17. The structure is contrasted with that of other cation-transporting antibiotics. Model building allows a structure that could stack to form a tunnel with a lipophilic exterior and hydrophilic interior and flexible internal arms formed by the pendant C-terminal glutamine residue.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

The primary structure of alamethicin

Payne

Jakes

Hartley

1970

Biochemical Journal

129

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“…Concentration-dependent investigations of the surface activity [ l l , 121, sedimentation measurements [ll], one 'H NMR study [12-161 and further conductivity measurements on artificial membranes [17 -231 show that alamethicin has a tendency to aggregate in neutral aequeous medium and that it might undergo hydrophobic interactions in phospholipids [12]. The tendency to form micelles at the lipid/water interface is also the basis of the models suggested so far to explain the molecular mechanism of the action of alamethicin on membranes [8,19].…”

Section: ' Rmentioning

confidence: 99%

“…CD, circular dichroism; NMR, nuclear magnetic resonance; Aib, a-amino-isobutyric acid; Yb(fod)3, 1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionato-ytterbium. .. show that alamethicin has a tendency to aggregate in neutral aequeous medium and that it might undergo hydrophobic interactions in phospholipids [12]. The tendency to form micelles at the lipid/water interface is also the basis of the models suggested so far to explain the molecular mechanism of the action of alamethicin on membranes [8,19].…”

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confidence: 99%

Conformational Changes of Alamethicin Induced by Solvent and Temperature

Jung

Dubischar

Leibfritz

1975

European Journal of Biochemistry

138

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I3C nuclear magnetic resonance (NMR) and circular dichroism (CD) have been used for studies on the conformation of alamethicin. The 13C NMR spectrum is assigned with the aid of signals of synthetic partial sequences and selective proton decoupling. The solvent and temperature-dependence of the I3C NMR spectra, TI measurements and the use of lanthanide-shift reagents allow the differentiation between the amino acids belonging to a rigid a-helical portion of the alamethicin sequence and those belonging to a more flexible part. The 13C NMR results are in agreement with results obtained from extended solvent and temperature-dependent CD studies which indicate a highly stabilized nonpolar and intrachenar a-helical part. The concentration-dependence of the CD spectrum of alamethicin in a nematic phase revealed aggregation phenomena which might simulate those observed in natural and synthetic membranes. After dissolving alamethicin in aqueous alcohol there is a time-dependence of the ellipticity of the Cotton effects showing a sort of memory effect on the mode of dissolution. Four different conformations can be characterized by CD spectra depending on the solvent and concentration. A model illustrating the dynamic conformations and aggregation phenomena within a membrane is proposed.Alamethicin, a membrane active peptide (antibiotic U-22324) can be isolated from the culture broth of the fungus Trichoderma viride [ 11. Biosynthetic studies [2] suggest a non-ribosomal enzymic mechanism for the synthesis of this unusual sequence. The verylipophilic polypeptide has only one ionized group in the form of a glutamine carboxylic group. In addition to a y-glutamyl peptide bond there are seven residues of a-aminoisobutyric acid which contribute to the characteristic properties of alamethicin. The primary structure of a cycle which comprises 17 amino acids was determined in 1970 [3] and confirmed by mass spectrometry [4].The peptide has strong lysing properties on membranes of Ehrlich ascites carcinoma cells and on erythrocytes. These appear within very narrow ranges Abbreviations. CD, circular dichroism; NMR, nuclear magnetic resonance; Aib, a-amino-isobutyric acid; Yb(fod)3, 1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedionato-ytterbium. .. show that alamethicin has a tendency to aggregate in neutral aequeous medium and that it might undergo hydrophobic interactions in phospholipids [12]. The tendency to form micelles at the lipid/water interface is also the basis of the models suggested so far to explain the molecular mechanism of the action of alamethicin on membranes [8,19]. The experiments exclude definitely a mobile carrier mechanism and point towards the formation of a hydrophilic ion-transporting "structure" in the membrane. So far ideas on the molecular structure of these membrane pores have been based on molecular model building. There have been no spectroscopic investigations on alamethicin, with the exception of a CD study [23,24] and a 60-MHz 'H NMR spectrum [13]. Therefore we have been carrying out conformatio...

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“…Despite a number of physicochemical (13,14), conformational (15)(16)(17)(18), and chemical (17)(18)(19)(20)(21)(22) studies, the primary structure of ALA remains unknown. Currently, there are two proposed sequences: a cyclic (ALA-c) and an open chain (ALA-o) peptide structure (Fig.…”

mentioning

confidence: 99%

Synthesis of a 19-residue peptide with alamethicin-like activity.

Gisin¹,

Kobayashi²,

Hall³

1977

Proc. Natl. Acad. Sci. U.S.A.

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This The peptide also affects the electrical properties of planar artificial lipid membranes in a unique and specific way: minute concentrations of ALA induce voltage-dependent conductances that mimic the action potential phenomena observed in nerve membranes. These effects were observed by Mueller and Rudin (6) and have been studied also by other groups (7-9). It is now generally believed that several ALA molecules aggregate in the membrane to form ion-conducting channels (8,(10)(11)(12).Despite a number of physicochemical (13, 14), conformational (15-18), and chemical (17-22) studies, the primary structure of ALA remains unknown. Currently, there are two proposed sequences: a cyclic (ALA-c) and an open chain (ALA-o) peptide structure (Fig. 1). The former, proposed by Payne et al. (19), has been confirmed by Ovchinnikov et al. (20), while the latter, proposed by Martin and Williams (21,22), found the support of the Jung group (17). The two proposals agree on a sequence of 15 consecutive residues (3-17 according to ALA-o numbering). In ALA-c the y-carboxyl group of Glu'8 is in amide linkage with Pro2 to form a cyclic structure. In

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Physical Studies of Phospholipid/Alamethicin Interactions

Cited by 38 publications

References 7 publications

The primary structure of alamethicin

The primary structure of alamethicin

Conformational Changes of Alamethicin Induced by Solvent and Temperature

Synthesis of a 19-residue peptide with alamethicin-like activity.

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