Membrane fusion induced by a short fusogenic peptide is assessed by its insertion and orientation into target bilayers.

To clarify the molecular mechanism by which an amphipathic negatively charged peptide consisting of 11 residues (WAE) induces fusion, and the relevance of these features for fusion, its mode of insertion and orientation into target bilayers were investigated. Using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) in combination with techniques based on tryptophan fluorescence, the peptide was found to form an alpha-helix, shallowly inserted into the membrane to which it is anchored. Interestingly, in the presence of target membranes, WAE inserts into the target bilayer as an alpha-helix oriented almost parallel to the lipid acyl chains. The accessibility of the peptide to either acrylamide (as an aqueous quencher of Trp fluorescence) or deuterium oxide (on the course of an FTIR deuteration kinetics) was lower in the presence than in the absence of target membranes, confirming that under those conditions, the peptide was shielded from the aqueous environment. Since fusion experiments have shown a temperature dependence, the effect of this later parameter on the structure and mode of insertion of the peptide was also analyzed. In the presence of target membrane, but not in their absence, the amount of alpha-helical structure increased with temperature, reflecting a similar temperature-dependent increase in the rate and extent of WAE-induced fusion. Also, the extent of penetration of the helix into the target membrane was greater at 37 degrees C than at lower temperatures. This temperature-dependent distinction was revealed by a decreased accessibility of the peptide to deuterium oxide and acrylamide at 37 degrees C as compared to that at lower temperatures. These data underscore the role of peptide structure, peptide penetration, and orientation in the mechanism of protein-induced membrane fusion.