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

[Image: see text] We have developed a model system for membrane fusion that utilizes lipidated derivatives of a heterodimeric coiled-coil pair dubbed E(3) (EIAALEK)(3) and K(3) (KIAALKE)(3). 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(3) and K(3) differently. We conclude that negatively charged E(3) acts as a “handle” for positively charged K(3) and facilitates liposome docking, the first stage of the fusion process, through coiled-coil formation. The efficacy of this E(3) handle is enhanced by longer spacer lengths. K(3) directs the fusion process via peptide–membrane interactions, but the length of the PEG spacer plays two competing roles: a PEG(4)/PEG(8) spacer length is optimal for membrane destabilization; however, a PEG(12) 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(3)–membrane interactions and thus mediates fusion more efficiently.