Two peptides, Coil-K and Coil-E, form a parallel heterodimeric coiled coil, CC-K/E, and have been shown to promote membrane fusion. This article examines the effects of reversing the sequence of Coil-E (to yield Coil-Er), on coiled-coil formation and membrane fusion. Coiled-coil assembly was studied using circular dichroism spectroscopy, paramagnetic proton NMR, fluorescence spectroscopy, analytical ultracentrifugation and computational simulations. Combined, the data show that Coil-K and Coil-Er combine in a 1 : 1 ratio to form an antiparallel tetramer, reinforcing previous studies that show small changes to peptide sequences strongly affect the stoichiometry and orientation of the resulting assemblies. Cholesterol-modified Coil-K and Coil-Er variants were subsequently tested for their ability to promote membrane fusion and the results were compared to the CC-K/E model system. Surprisingly, no significant differences were found between the two systems, despite the Coil-K/Er complex being ‘non-zipper-like’.