pardaxin and fluorexon

Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning confocal microscopy and natural abundance (13)C solid-state nuclear magnetic resonance to provide a more complete picture of how pardaxin interacts with lipids. The kinetics and efficiency of release of entrapped calcein is highly sensitive to pH. In vesicles containing zwitterionic lipids (PC), release occurs most rapidly at low pH, whereas in vesicles containing 20% anionic lipid (PG), release occurs most rapidly at high pH. Pardaxin forms stable or transient pores in PC vesicles that allow release of contents without loss of vesicle integrity, whereas the inclusion of PG promotes total vesicle collapse. In agreement with this, solid-state nuclear magnetic resonance reveals that pardaxin takes up a trans-membrane orientation in 14-O-PC/6-O-PC bicelles, whereas the inclusion of 14-0-PG restricts it to contacts with lipid headgroups, promoting membrane lysis. Pore formation in zwitterionic vesicles is more efficient than lysis of anionic vesicles, suggesting that electrostatic interactions may trap pardaxin in several suboptimal interconverting conformations on the membrane surface.

Topics: Amino Acid Sequence; Fish Venoms; Fluoresceins; Hydrogen-Ion Concentration; Kinetics; Lipid Metabolism; Lipids; Magnetic Resonance Spectroscopy; Microscopy, Confocal; Molecular Sequence Data; Permeability; Porosity; Protein Conformation; Protons; Unilamellar Liposomes

The mechanism of leakage induced by surface active peptides is not yet fully understood. To gain insight into the molecular events underlying this process, the leakage induced by the peptide pardaxin from phosphatidylcholine/ phosphatidylserine/cholesterol large unilamellar vesicles was studied by monitoring the rate and extent of dye release and by theoretical modeling. The leakage occurred by an all-or-none mechanism: vesicles either leaked or retained all of their contents. We further developed a mathematical model that includes the assumption that certain peptides become incorporated into the vesicle bilayer and aggregate to form a pore. The current experimental results can be explained by the model only if the surface aggregation of the peptide is reversible. Considering this reversibility, the model can explain the final extents of calcein leakage for lipid/peptide ratios of > 2000:1 to 25:1 by assuming that only a fraction of the bound peptide forms pores consisting of M = 6 +/- 3 peptides. Interestingly, less leakage occurred at 43 degrees C, than at 30 degrees C, although peptide partitioning into the bilayer was enhanced upon elevation of the temperature. We deduced that the increased leakage at 30 degrees C was due to an increase in the extent of reversible surface aggregation at the lower temperature. Experiments employing fluorescein-labeled pardaxin demonstrated reversible aggregation of the peptide in suspension and within the membrane, and exchange of the peptide between liposomes. In summary, our experimental and theoretical results support reversible surface aggregation as the mechanism of pore formation by pardaxin.

Topics: Amino Acid Sequence; Biophysical Phenomena; Biophysics; Cholesterol; Fish Venoms; Fluoresceins; Fluorescent Dyes; Kinetics; Liposomes; Models, Chemical; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylserines; Surface Properties; Temperature

Synthetic calmodulin-binding (CaM-binding) peptides (CBPs) representing CaM-binding domains of Ca2+/CaM-dependent enzymes have been reported to interfere with the activity of the melanocyte-stimulating hormone (MSH) receptor function in melanoma cells [Gerst, J. E., & Salomon, Y. (1988) J. Biol. Chem. 263, 7073-7078]. We postulated that membrane lipids may play an important role in the mode of action of CBPs on cells. We therefore tested the ability of CBPs to interact with membrane bilayers. Using artificial phospholipid vesicles, or M2R melanoma cells and cell membranes derived therefrom, as models, we report here that synthetic peptides representing the CaM-binding domains of skeletal muscle myosin light chain kinase (M5) and the human erythrocyte calcium pump (C28W), as well as other CBPs, interact with lipid bilayers and cell membranes. Significant interactions of CBPs with the lipid bilayer were detected in both model systems. M5 and C28W were found to partition into the lipid bilayer of melanoma cell membranes and soybean lecithin vesicles, and surface partition constants obtained (for the liposome model) were in the range 10(3)-10(4) M-1. In addition, C28W and its N-modified NBD derivative were found to inhibit [125I]iodo-[Nle4,D-Phe7]alpha MSH binding to cultured M2R melanoma cells. These and other CBPs were also found to induce the release of cations and calcein from liposomes, suggesting that the interaction of CBPs with the lipid bilayer increases membrane permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha-MSH; Amino Acid Sequence; Animals; Binding Sites; Calcium-Transporting ATPases; Calmodulin; Calmodulin-Binding Proteins; Cell Membrane; Cell Membrane Permeability; Erythrocytes; Fish Venoms; Fluoresceins; Humans; Lipid Bilayers; Melanoma, Experimental; Melitten; Mice; Molecular Sequence Data; Muscles; Myosin-Light-Chain Kinase; Peptides; Tumor Cells, Cultured

The influence of specific L- to D-amino acid substitutions on the interaction of pardaxin, a shark repellent neurotoxin polypeptide, with phospholipid vesicles and human erythrocytes is described. Twelve modified, truncated, or fluorescently labeled [with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD) at their N-terminal amino acid] analogues of pardaxin were synthesized by a solid-phase method. Fluorescence measurements were used to monitor the interaction of the analogues with membranes [Rapaport, D., & Shai, Y. (1991) J. Biol. Chem. 266, 23769-23775]. Upon titration of solutions containing the NBD-labeled peptides with small unilamellar vesicles, the fluorescent emission spectra of all NBD-labeled peptides displayed similar blue-shifts, in addition to enhanced intensities, upon relocation of the probe to the more apolar environment. Binding isotherms were constructed from which surface partition constants, in the range of 10(4) M-1, were derived. The existence of an aggregation process, suggested by the shape of the binding isotherms, could be associated only with those analogues in which the N-helix (residues 1-9) was not perturbed. The alpha-helical content of the analogues was estimated by circular dichroism (CD) spectroscopy, both before and after binding to vesicles at neutral pH. The ability of the peptides to dissipate a diffusion potential and to cause calcein release, as well as to lyse human erythrocytes, served to functionally characterize the peptides. The results support a two alpha-helix model, with a bend at position 13, as best describing pardaxin in its membrane-bound state.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acids; Cell Membrane Permeability; Circular Dichroism; Diffusion; Erythrocyte Membrane; Fish Venoms; Fluoresceins; Fluorescence; Fluorescent Dyes; Humans; Liposomes; Proline; Protein Structure, Secondary; Solubility; Stereoisomerism; Valinomycin

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.

Topics: Amino Acid Sequence; Cell Survival; Erythrocytes; Fish Venoms; Fluoresceins; Humans; Indicators and Reagents; Ion Channels; Lipid Bilayers; Membrane Potentials; Molecular Sequence Data; Peptides; Structure-Activity Relationship