Channel formation properties of synthetic pardaxin and analogues.
Six analogues of teh 33-residue shark repellent neurotoxin pardaxin were synthesized by the solid phase method: [Ala13]pardaxin, [Gly14,Gly15]pardaxin, des[1----9]pardaxin, [N1-succinamido]pardaxin, C33-dihydroxyethylamido]pardaxin, and C33-[diaminoethylamido]pardaxin. The spectroscopic and functional characterizations of the analogues are described. The peptides were characterized spectroscopically by circular dichroism (CD) before and after binding to soybean vesicles. They were characterized functionally by measuring their potential to evoke the dissipation of diffusion potential and calcein release from sonicated unilamellar soybean liposomes, by determining their ability to create single channels in planar bilayers, and by measuring their cytolytic activity on human erythrocytes. The behavior of the analogues modified at the C terminus is similar to that of pardaxin. [N'-succinamido]Pardaxin, however, reveals an increase in alpha-helicity both alone and in the presence of liposomes. It has the same potency as pardaxin to dissipate diffusion potential, to evoke calcein release and to produce single channels in lipid bilayers, but at a slower rate than that of pardaxin. It has more than 70-fold less cytolytic activity than pardaxin. [Ala13] Pardaxin has twice the alpha-helical content than pardaxin, both alone and in the presence of vesicles, yet it has less effect on the diffusion potential and calcein release, and it does not have cytolytic activity on human erythrocytes. Both [Gly14,Gly15]pardaxin and des[1----9]pardaxin are much less potent than pardaxin in all effects. However des[1----9]pardaxin exhibits a slight change in alpha-helicity upon binding to vesicles, whereas [Gly14,Gly15]pardaxin does not. The results support a model in which pardaxin is composed of two putative alpha-helices separated by proline. The N-terminal alpha-helix is important for the insertion of the peptide to the lipid bilayer, and the C-terminal amphiphilic alpha-helix is the ion channel lining segment of pardaxin.
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Y
Y
Shai
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Bach
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Yanovsky
D000595:Amino Acid Sequence; D002470:Cell Survival; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects; D005397:Fish Venoms / Q000378:metabolism* / Q000494:pharmacology; D005452:Fluoresceins; D006801:Humans; D007202:Indicators and Reagents; D007473:Ion Channels; D008051:Lipid Bilayers; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis*; D013329:Structure-Activity Relationship
D005397:Fish Venoms; D005452:Fluoresceins; D007202:Indicators and Reagents; D007473:Ion Channels; D008051:Lipid Bilayers; D010455:Peptides; C017603:pardaxin; C007740:fluorexon