The pH-dependent bilayer destabilization by an amphipathic peptide

Abstract

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)

On the formulation of pH-sensitive liposomes with long circulation times

“…The levels of Transfectam (lipopolyamine)-mediated transfection of H225 human melanoma cells could be increased by up to 1000-fold (over that obtained with a sub-optimal charge-equivalent (1.5) of Transfectam/DNA) by adding the hemagglutinin-derived peptide INF6 to the preformed lipoplexes [68]. The association of the pH-sensitive peptide GALA [69,70] with DOTAP/DOPE (1:1) liposomes before complexation with plasmid DNA resulted in a significant enhancement in luciferase expression in COS-7 cells, which depended on the cationic liposome/DNA (+/ À ) charge ratio [71].…”

Non-viral gene delivery in skeletal muscle: a protein factory

“…83 This peptide, GALA (WEAALAEALAEA-LAEHLAEALAEALEALAAGGSC), has repeat units of glutamic acid-alanine-leucine-alanine and possesses the property of destabilizing membranes in a pH-dependent manner. Parente et al examined the membrane-destabilizing effect of GALA peptide, on the experimental model of lipid biolayer vesicles of egg phosphatidylcholine in a lipid/peptide molar ratio of 500/1.…”

Transfection of human macrophages by lipoplexes via the combined use of transferrin and pH-sensitive peptides

“…1). GALA undergoes a transition from a random coil at pH 7.5 to an amphipathic ␣-helix at pH 5.0, under which conditions it strongly interacts with phospholipid membranes to induce fusion, contents leakage, and phospholipid flip-flop [17,29,34,35]. The covalent attachment of GALA to polyamidoamine dendrimers was found previously to enhance transfection of cell lines by 1:1 (terminal amine: phosphate) complexes by several orders of magnitude [36].…”

“…The GALA peptide [17,29] and the peptide named HA-2, derived from the amino-terminal sequence of the influenza virus hemagglutinin subunit HA2 [18] were synthesized and high-performance liquid chromatography (HPLC)-purified by the UCSF Biomolecular Resource Center. GALA is a 30-amino acid, pHsensitive, amphipathic peptide with the sequence WEAALAEALAEA-LAEHLAEALAEALEALAA.…”