Fig. 1: Binding of IM30 to negatively charged membrane surfaces results in ring disassembly, carpet formation, and membrane stabilization.

a FRET was measured using IM30 rings containing both, IM30-CFP and IM30-Venus monomers (red). The normalized relative fluorescence intensity (see “Methods”) is plotted against the DOPG concentration. The intensity decreases with increasing DOPG concentrations, indicating an increasing average distance between the monomers upon interaction with membranes. Noteworthy, the fluorescence characteristics of the fluorophores alone change only slightly upon membrane binding, resulting in an apparently altered FRET (black). The error bars represent SD, n = 5. b The structure of IM30 WT and IM30* bound on a PG bilayer was imaged via AFM (the false-color ruler indicates the heights in the images). Both IM30 variants form carpet-like structures. The height-profiles (white section lines in the images) of the carpet-like structures indicate similar heights of IM30 WT (black line) and IM30* (red line) carpets. Determined heights are in the range of 0.7–1.9 nm). Single coherent IM30* carpets have increased dimensions, which leads to edges appearing rounder than the fractal-like shape of IM30 WT carpets. Scale bar: 1 µm (upper panel) and 3 µm (lower panel). c IM30 appears to initially bind to the membrane as a ring, followed by disassembly into small oligomers/monomers and rearrangement to a carpet-like structure. The ring structure was taken from EMD:3740¹⁹. d ACMA fluorescence was used to monitor proton flux across DOPG membranes. Untreated liposomes were slightly permeable for protons (positive control, dark gray), whereas DOPG liposomes have high proton permeability in presence of 6% isopropanol (negative control, light gray). Lysozyme, which was used as a control (cyan), had no effect on the proton permeability. In presence of IM30 WT (black), the proton permeability of isopropanol-treated DOPG liposomes was reduced. This effect was much stronger in presence of IM30* (red). For quantitative analysis, the initial slope of the fluorescence changes was evaluated. Error bars represent SD (n = 3). e Lipid-binding of IM30 WT (black) and IM30* (red) to PG liposomes was determined via monitoring Laurdan fluorescence changes. IM30* affects the Laurdan fluorescence emission characteristics (ΔGP) much faster than the WT protein. Error bars represent SD (n = 3).