melitten and 6-carboxyfluorescein

We have investigated, both experimentally and theoretically, the efflux of carboxyfluorescein (a self-quenching fluorescent dye) from vesicles of different sizes and lipid species (POPC, DOPC) after having added the bee venom peptide melittin. This comprises quantitative analyses regarding the extent of lipid-associated peptide, the mode as well as the temporal progress of dye release and the possible leakage mechanism. Our results indicate a graded efflux characterized by a single-pore retention factor reflecting the formation of pores whose lifetimes are rather small (millisecond range). The observed fluorescence signal arising from the dequenching of effluent dye has been converted to the number of pore openings over the course of time. All the resulting curves exhibit a pronounced slowing down of the pore formation rate revealing two distinct relaxation steps at about 20 and 200 s, respectively, being largely independent of vesicle type and peptide to lipid ratio. The pore formation rate itself increases in proportion to the amount of membrane bound peptide. We give a quantitative account of our experimental findings based on a novel reaction scheme applicable to any of our various liposome systems. It implies that the pore formation rate is controlled by a passage through two intermediate monomeric peptide states. These states are thought to become well populated in the initial stage of lipid bilayer perturbation, but would practically die out after some time owing to a restabilization of the membrane system.

Topics: Binding Sites; Energy Transfer; Fluoresceins; Fluorescent Dyes; In Vitro Techniques; Kinetics; Liposomes; Melitten; Phosphatidylcholines; Spectrometry, Fluorescence

We investigated the interaction of the peptide melittin with differently sized vesicles consisting of various lipid compositions. This system was characterized by dynamic light scattering to estimate the size of vesicles. For SUV we obtained a radius of 12 nm, for LUV 53 nm. The pore forming process of melittin in vesicles was investigated by efflux of encapsulated fluorescent dyes at a self-quenching concentration. The influence of the following parameters on efflux and pore formation was estimated: lipid composition (POPC and DOPC), vesicle size (SUV and LUV) and size of the encapsulated dye (carboxyfluorescein and FITC-dextran). We found that under similar conditions vesicles of DOPC give always less leakage than vesicles of POPC independent of the fluorescent dye. For SUV and LUV we have obtained a different leakage behaviour at identical surface concentrations of melittin (if the same partition coefficient is assumed). From efflux measurements with different dyes we concluded that 6-20 molecules of melittin are necessary to form a pore. The possibility that not pore formation but fusion is the mechanism of melittin induced efflux was disproved by fusion experiments using a resonance energy transfer assay.

Topics: Amino Acid Sequence; Cell Membrane; Dextrans; Fluorescein-5-isothiocyanate; Fluoresceins; Light; Liposomes; Melitten; Membrane Fusion; Membrane Lipids; Membranes, Artificial; Molecular Sequence Data; Phosphatidylcholines; Scattering, Radiation

Interaction of melittin with lipid membranes was studied systematically with respect to its adsorption onto membranes, its effect on membrane leakage and fusion, and micellization at various melittin/lipid ratios. It was found that melittin has a strong affinity for adsorption onto lipid membranes. The analysis of the measured electrophoretic mobilities by use of a Gouy-Chapman double layer theory, shows that melittin is adsorbed onto the phosphatidylserine membrane several times more than the phosphatidylcholine membrane. However, it was observed that the phosphatidylcholine membrane is more susceptible to membrane leakage, vesicle fusion and micellization at a lower level of melittin adsorbed than the phosphatidylserine membrane. For small unilamellar phosphatidylcholine vesicles in 0.1 M NaCl, membrane leakage started at melittin to lipid ratio of 1:2000, a large increase in the rate of membrane leakage occurred at a ratio of about 1:500 or higher, membrane fusion occurred at a ratio of 1:200, and membrane micellization at a ratio of 1:10. On the other hand, for small unilamellar phosphatidylserine vesicles, the respective concentrations of melittin to result in membrane leakage, vesicle fusion, and membrane micellization were several times higher. Surface pressure measurements of lipid monolayers showed that the increase in surface pressure of the phosphatidylcholine monolayer due to the presence of melittin in the subphase solution was greater than that for the phosphatidylserine monolayer at any melittin concentration in the subphase solution. These experimental results indicate that melittin tends to be adsorbed on the surface of the negatively charged phosphatidylserine membrane due to the electrostatic binding so that the melittin molecule can stay out more on the surface of the membrane, while melittin appears to be adsorbed more into the hydrophobic membrane core for the electrically neutral phosphatidylcholine membrane.

Topics: Adsorption; Fluoresceins; Lipid Bilayers; Lysophospholipids; Mathematics; Melitten; Membrane Fusion; Micelles; Particle Size; Permeability

Melittin fragments carrying spiropyran were synthesized, and their distribution in phospholipid bilayer membrane was studied by using spiropyran as a probe. Spiropyran was connected to the side chain of a Glu residue (Glu(OSp)), and the residue was replaced for the fourth position of melittin (1-7) fragment (M7Sp). M7Sp showed a high affinity for phospholipid membrane. The spiropyran group of M7Sp was converted to a merocyanin group by UV irradiation, which reduced the amount of the peptide bound to the membrane to the half of the initial amount. The location of the merocyanin group of M7Sp in the membrane was evaluated by the rate of thermal isomerization from merocyanin to spiropyran, which is sensitive to the microenvironment of merocyanin. A large fraction of the merocyanin group isomerized rapidly back to a spiropyran form, indicating that M7Sp is located in a relatively hydrophobic region of the membrane. Although the interaction of the peptide with phospholipid membrane is affected by photoisomerization of the spiropyran substituent, spiropyran was shown to be a useful tool to evaluate the location of the peptide in the lipid membrane.

Topics: Amino Acid Sequence; Benzopyrans; Chemical Phenomena; Chemistry, Physical; Fluoresceins; Fluorescent Dyes; Indoles; Isomerism; Kinetics; Melitten; Membranes, Artificial; Molecular Sequence Data; Nitro Compounds; Peptide Fragments; Phospholipids; Pyrimidinones; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Thermodynamics

The cationic peptide antibiotics Pep 5, nisin and subtilin depolarize bacterial and artificial membranes by formation of voltage-dependent multi-state pores. Studies with non-energized liposomes indicated that the peptides do not span the membrane in the absence of a membrane potential. The effects of Pep 5 and nisin on neutral membranes, as studied by membrane fluidity, phase transition points and carboxyfluorescein efflux, were small compared to melittin. Acidic liposomes were affected more strongly, indicative of primarily electrostatic interactions with phospholipid head groups. Subtilin may slightly enter the hydrophobic core as suggested by tryptophan fluorescence quenching and liposome fusion experiments.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Bacteriocins; Cell Membrane; Fluoresceins; Liposomes; Melitten; Membrane Fluidity; Membrane Fusion; Membrane Potentials; Nisin; Peptides; Peptides, Cyclic; Spectrometry, Fluorescence; Temperature; Tryptophan