gala-peptide and 1-palmitoyl-2-oleoylphosphatidylcholine

GALA is a 30 residue synthetic peptide designed to interact with membranes in a pH-sensitive manner, with potential applications for intracellular drug and gene delivery. Upon reduction of the pH from neutral to acidic, GALA switches from random coil to alpha-helix, inserts into lipid bilayers, and forms oligomeric pores of defined size. Its simple sequence and well-characterized behavior make the peptide an excellent starting point to explore the effects of sequence on structure, pH sensitivity, and membrane affinity. We describe synthesis and characterization of two derivatives of GALA, termed GALAdel3E and YALA. GALAdel3E has a deletion of three centrally located glutamate residues from GALA, while YALA replaces one glutamate residue with the unusual amino acid 3,5-diiodotyrosine. Both derived peptides retain pH sensitivity, showing no ability to cause leakage of an encapsulated dye from unilamellar vesicles at pH 7.4 but substantial activity at pH 5. Unlike GALA, neither peptide undergoes a conformational change upon reduction of the pH, remaining alpha-helical throughout. Interestingly, the pH at which the peptides activate is shifted, with GALA becoming active at pH approximately 5.7, GALAdel3E at pH approximately 6.2, and YALA at pH approximately 6.7. Furthermore, the peptides GALAdel3E and YALA show improved activity compared with GALA for cholesterol-containing membranes, with YALA retaining the greatest activity. Improved activity in the presence of cholesterol and onset of activity in the critical range between pH 6 and 7 may make these peptides useful in applications requiring intracellular delivery of macromolecules, such as gene delivery or anti-cancer treatments.

Topics: Amino Acid Sequence; Cholesterol; Circular Dichroism; Diiodotyrosine; Fluoresceins; Hydrogen-Ion Concentration; Liposomes; Membranes; Molecular Sequence Data; Peptides; Phosphatidylcholines; Porins; Protein Structure, Secondary

We determined the orientation of a biotinylated version of the pore-forming peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) at pH 5.0 in large unilamellar phosphatidylcholine vesicles, using the enhancement of BODIPY-avidin fluorescence subsequent to its irreversible binding to a biotin moiety. GALA and its variants were biotinylated at the N- or C-terminus. BODIPY-avidin was either added externally or was pre-encapsulated in vesicles to assess the fraction of liposome-bound biotinylated GALA that exposed its labeled terminus to the external or internal side of the bilayer, respectively. Under conditions where most of the membrane-bound peptides were involved in transmembrane aggregates and formed aqueous pores (at a lipid/bound peptide molar ratio of 2500/1), the head-to-tail (N- to C-terminus) orientation of the membrane-inserted peptides was such that 3/4 of the peptides exposed their N-terminus on the inside of the vesicle and their C-terminus on the outside. Under conditions resulting in reduced pore formation (at higher lipid/peptide molar ratios), we observed an increase in the fraction of GALA termini exposed to the outside of the vesicle. These results are consistent with a model (Parente et al., Biochemistry, 29:8720, 1990) that requires a critical number of peptides (M) in an aggregate to form a transbilayer structure. When the peptides form an aggregate of size i, with i < M = 4 to 6, the orientation of the peptides is mostly parallel to the membrane surface, such that both termini of the biotinylated peptide are exposed to external BODIPY-avidin. This BODIPY-avidin/biotin binding assay should be useful to determine the orientation of other membrane-interacting molecules.

Topics: Amino Acid Sequence; Avidin; Biophysical Phenomena; Biophysics; Biotin; Boron Compounds; Fluorescent Dyes; Hydrogen-Ion Concentration; Lipid Bilayers; Liposomes; Peptides; Phosphatidylcholines; Protein Binding

The effect of cholesterol on the bilayer partitioning of the peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) and its assembly into a pore in large unilamellar vesicles composed of neutral and negatively charged phospholipids has been determined. GALA undergoes a conformational change from a random coil to an amphipathic alpha-helix when the pH is reduced from 7.0 to 5.0, inducing at low pH leakage of contents from vesicles. Leakage from neutral or negatively charged vesicles at pH 5.0 was similar and could be adequately explained by the mathematical model (Parente, R. A., S. Nir, and F. C. Szoka, Jr., 1990. Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA. Biochemistry. 29:8720-8728) which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of 10 +/- 2 peptides. Increasing cholesterol content in the membranes resulted in a reduced efficiency of the peptide to induce leakage. Part of the cholesterol effect was due to reduced binding of the peptide to cholesterol-containing membranes. An additional effect of cholesterol was to increase reversibility of surface aggregation of the peptide in the membrane. Results could be explained and predicted with a model that retains the same pore size, i.e., 10 +/- 2 peptides, but includes reversible aggregation of the monomers to form the pore. Resonance energy transfer experiments using fluorescently labeled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol-containing liposomes, thus reducing the efficiency of pore formation.

Topics: Amino Acid Sequence; Cholesterol; Dextrans; Hydrogen-Ion Concentration; Kinetics; Lipid Bilayers; Models, Biological; Models, Theoretical; Molecular Sequence Data; Peptides; Phosphatidylcholines; Phosphatidylglycerols; Protein Conformation; Protein Structure, Secondary

GALA, a synthetic, amphipathic 30-amino-acid peptide, based upon a Glu-Ala-Leu-Ala motive, was designed to mimic the behavior of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH, and becomes an amphipathic alpha helix as the pH is lowered to 5, where it interacts with phospholipid bilayers. Attenuated total-reflection infrared spectroscopy, using polarized light, provides information on the structure and orientation of the peptide and the lipids, which is not subject to artifacts due to light scattering with large particles. H/2H-exchange rate of the amide N-H group and analysis of the shape of the amide I' by Fourier self-deconvolution and curve fitting indicate that the alpha-helical content increases from 19% to 69%, on lowering the pH. A further increase to 100% alpha helix is observed after interaction with palmitoyloleoylglycerophosphocholine (PamOleGroPCho) vesicles. Dichroism data obtained with oriented bilayers of the PamOleGroPCho-GALA complex demonstrate that PamOleGroPCho hydrocarbon chains and the peptide alpha helical axis are essentially perpendicular (+/- 15) to the membrane plane. At neutral pH, in the presence of dimyristoylglycerophosphocholine (Myr2GroPCho), GALA is known to form discoidal structures similar to those formed under the same conditions by apolipoproteins AI and AII. In these discoidal complexes, the alpha-helical content was estimated to be 65%, with the rest of the structure being essentially unordered. No significant modification of the all-trans conformation of the hydrocarbon chain of Myr2GroPCho was detected upon disc formation. Dichroism measurements show that the alpha-helical axis is essentially parallel to the hydrocarbon chains. These data support a model in which a discoidal patch of the bilayer is surrounded by amphipathic helices which shield the hydrophobic region of the bilayer from the aqueous environment. The infrared spectrum of GALA in this complex was found to be very similar to those of apolipoproteins AI and AII which form discoidal complexes with Myr2GroPCho, but the spectrum is quite different from that of apolipoprotein B100 in low-density lipoproteins, which does not form discoidal complexes.

Topics: Amino Acid Sequence; Apolipoproteins; Dimyristoylphosphatidylcholine; Lipid Bilayers; Molecular Sequence Data; Peptides; Phosphatidylcholines; Protein Conformation; Solubility; Spectrophotometry, Infrared