Membrane-Fusogen Distance Is Critical for Efficient Coiled-Coil-Peptide-Mediated Liposome Fusion.

We have developed a model system for membrane fusion that utilizes lipidated derivatives of a heterodimeric coiled-coil pair dubbed E (EIAALEK) and K (KIAALKE). In this system, peptides are conjugated to a lipid anchor via a poly(ethylene glycol) (PEG) spacer, and this contribution studies the influence of the PEG spacer length, coupled with the type of lipid anchor, on liposome-liposome fusion. The effects of these modifications on peptide secondary structure, their interactions with liposomes, and their ability to mediate fusion were studied using a variety of different content mixing experiments and CD spectroscopy. Our results demonstrate the asymmetric role of the peptides in the fusion process because alterations to the PEG spacer length affect E and K differently. We conclude that negatively charged E acts as a "handle" for positively charged K and facilitates liposome docking, the first stage of the fusion process, through coiled-coil formation. The efficacy of this E handle is enhanced by longer spacer lengths. K directs the fusion process via peptide-membrane interactions, but the length of the PEG spacer plays two competing roles: a PEG/PEG spacer length is optimal for membrane destabilization; however, a PEG spacer increases the fusion efficiency over time by improving the peptide accessibility for successive fusion events. Both the anchor type and spacer length affect the peptide structure; a cholesterol anchor appears to enhance K-membrane interactions and thus mediates fusion more efficiently.