pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide.
A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences 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 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.
D000595:Amino Acid Sequence; D003911:Dextrans; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008081:Liposomes; D010455:Peptides; D010713:Phosphatidylcholines; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan
D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010455:Peptides; D010713:Phosphatidylcholines; C015219:fluorescein isothiocyanate dextran; D014364:Tryptophan; D016650:Fluorescein-5-isothiocyanate