Figure 3: Helix formation by the amphipathic helical (AH) motif requires membrane defects.

(a) CD spectra of 100 μM WT and LV/EE full-length complexin in the absence/presence of SUVs and LUVs. Increased helicity is observed only for the WT protein with SUVs. (b) Comparison of membrane binding, as observed by NMR (circles), with helix formation, as observed by the CD signal at 222 nm (squares), at increasing SUV concentrations with 100 μM full-length WT complexin. [θ]₂₂₂ was used to estimate the number of helical residues. Individual spectra shown in the inset (0–20 mM lipid from black to light grey). (c) Helix formation by the isolated complexin CTD (residues 91–143) plotted as the number of helical residues estimated using [θ]₂₂₂, (see Methods) at increasing concentrations of: 85/15 POPC/POPS LUVs (green triangles), 85/15 POPC/POPS SUVs (black squares), 60/25/15 DOPC/DOPE/DOPS LUVs (orange circles), 60/25/15 DOPC/DOPE/DOPS SUVs (cyan triangles), 55/30/15 POPC/POPE/POPS LUVs (red stars) and 55/30/15 POPC/POPE/POPS SUVs (blue diamonds). Helix formation occurs only for SUVs or for LUVs containing dioleoyl lipids. (d) Schematic model for saturable helix formation by the AH motif upon addition of SUVs. SUVs are depicted with multiple small defects capable of binding disordered protein regions and a single larger defect capable of binding an AH. Note that defects likely form dynamically⁴¹, and should not be considered as stable features. As noted in the text, it appears that highly curved vesicles can form/accommodate a limited number of defects extensive enough to accommodate helix formation by the AH motif. At the lowest depicted SUV concentration, most of the protein (two out of four depicted molecules) is bound to the smaller defects in a disordered conformation and only a small amount (one out of four molecules) can bind in a helical conformation, whereas the remainder (one out of four molecules) remains unbound. At double the SUV concentration, all of the protein can bind, but only half (two of four depicted molecules) can bind in a helical conformation, whereas the other half can only bind in a disordered conformation. Doubling the SUV concentration again allows all of the protein molecules to bind in a helical conformation.