The synthetic, amphipathic peptide GALA undergoes a pH-dependent conformational change and induces leakage of contents from large unilamellar phosphatidylcholine vesicles when in a helical conformation. The kinetics of this process have been investigated over a wide range of pH and lipid and peptide concentrations. Leakage from lipid vesicles is rapidly initiated (within 2 s) when the pH is lowered below 6 and is rapidly terminated when the pH is raised to 7.5. The leakage shows a selectivity to the size of the entrapped molecules and occurs by an all or none mechanism; vesicles either leak or retain all of their contents. Using this experimental data, we have developed a mathematical description of the kinetics of leakage induced by GALA. This model assumes that GALA becomes incorporated into the vesicle bilayer and aggregates to form a pore. Leakage occurs when a critical number of peptides assemble into a supramolecular aggregate in the bilayer. Leakage curves generated at lipid/peptide ratios ranging from 500/1 to 30000/1 can be well described by this formalism. On the basis of the results and the model, we suggest that GALA forms a transbilayer channel composed of 8-12 monomers. The channel diameter ranges from 5 to 10 A. To the best of our knowledge, this is the first model that can predict the leakage kinetics of solutes entrapped in lipid vesicles induced by a pore-forming peptide. The analysis may be of general use in defining the kinetics and state of aggregation of similarly acting peptides and proteins which form multimeric assemblies in membranes.
A 30-residue amphipathic peptide was designed to interact with uncharged bilayers in a pH-dependent fashion. This was achieved by a pH-induced random coil-alpha-helical transition, exposing a hydrophobic face in the peptide. The repeat unit of the peptide, glutamic acid-alanine-leucine-alanine (GALA), positioned glutamic acid residues on the same face of the helix, and at pH 7.5, charge repulsion between aligned Glu destabilized the helix. A tryptophan was included at the N-terminal as a fluorescence probe. The rate and extent of peptide-induced leakage of contents from large, unilamellar vesicles composed of egg phosphatidylcholine were dependent on pH. At pH 5.0 with a lipid/peptide mole ratio of 500/1, 100% leakage of vesicle contents occurred within 1 min. However, no leakage of vesicle contents occurred at pH 7.5. Circular dichroism measurements indicated that the molar ellipticity at 222 nm changed from about -4000 deg cm2 dmol-1 at pH 7.6 to -11,500 deg cm2 dmol-1 at pH 5.1, indicating a substantial increase in helical content as the pH was reduced. Changes in molar ellipticity were most significant over the same pH range where a maximum change in the extent and rate of leakage occurred. The tryptophan fluorescence emission spectra and the circular dichroism spectra of the peptide, in the presence of lipid, suggest that GALA did not associate with the bilayer at neutral pH. A change in the circular dichroism spectrum and a blue shift of the maximum of the tryptophan fluorescence emission spectra at pH 5.0, in the presence of lipid, indicated an association of GALA with the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)
A kinetic model for pore-mediated and perturbation-mediated flip-flop is presented and used to characterize the mechanism of peptide-induced phospholipid flip-flop in bilayers. The model assumes that certain peptides can bind to and aggregate within the membrane. When the aggregate attains a critical size (M peptides), a channel is created that results in a fast flip-flop of phospholipids. In addition, certain peptides induce flip-flop through perturbation of the membrane without forming a pore. Donor phospholipid vesicles with an asymmetrical distribution of the fluorescent phospholipid 1-oleoyl-2-[12-[(7-nitro-1,2,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) were used to measure the extent of flip-flop by quantitating the decrease in fluorescence as the NBD-PC exchanged from the donor vesicles to acceptor vesicles that contained a quencher of the NBD fluorescence. Flip-flop curves generated at lipid/peptide ratios ranging from 30/1 to 300000/1 could be well-simulated by the model. Pore-forming peptides, such as melittin or the synthetic peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA), induce rapid phospholipid flip-flop with half-times for flip-flop of seconds at low peptide/vesicle ratios. The deduced pore sizes are M = 10 +/- 2 for GALA and M = 2 - 4 for melittin. The synthetic peptide LAGA (WEAALAEAEALALAEHEALALAEAELALAA) can catalyze flip-flop via bilayer perturbation. In contrast, hydrophobic peptides such as gramicidin A and valinomycin intercalate into the membrane, but induce little flip-flop. Modeling of the kinetics of phospholipid translocation supports pore formation as the key factor in accelerating phospholipid flip-flop. Thus, amphipathic segments from membrane proteins may account for non-energy-dependent phospholipid flip-flop in biological membranes.
The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.
Large unilamellar vesicles (LUV) have been prepared by three procedures from several synthetic and natural phosphatidylcholines. Reverse-phase evaporation vesicles (REV) and fusion vesicles were prepared by established procedures. A published procedure for the preparation of dialyzed octyl glucoside vesicles (DOV) was modified to allow its use with synthetic phospholipids. Negative-staining and freeze--fracture electron microscopy was used to determine the vesicle size distribution (mean diameters 800-1000 A) and extent of oligolamellar contamination in DOV preparations. Trapping of 6-carboxyfluorescein yielded measurements of the internal volume (2.6 +/- 0.3 microL/mumol of Pi) consistent with the size distributions determined by electron microscopy. An upper limit of less than 3 mol % oligolamellar vesicle contamination was indicated by calorimetric heat capacity profiles. The phase behaviors of large multilamellar vesicles and all three types of LUV were compared by using high-sensitivity differential scanning calorimetry and fluorescence depolarization of the membrane probe diphenylhexatriene. The most remarkable feature was the increased breadth of the main transition of DOV and of REV relative to the multilamellar species and to fusion vesicles. Both the main transition and the pretransition occurred at nearly the same temperatures in unilamellar and multilamellar species, but the unilamellar pretransition involved less than half the enthalpy observed in the multilamellar transition. Additional experiments indicated that the broadened main phase transition associated with DOV and REV reflected bilayer impurities resulting from preparation. It is concluded that LUV prepared by procedures that avoid impurities undergo a highly cooperative phase transition, as demonstrated here for fusion vesicles.
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