Figure 1: IM30 interaction with membrane lipids.

SD gradient flotation analysis of (a) native IM30 attached to Synechocystis membranes, (b) heterologously expressed IM30 added to urea-washed membranes and (c) solely heterologously expressed IM30. Samples were separated on 34–68% SD gradients, fractionated and analysed by SDS–PAGE and western blot analyses, using an anti-IM30 antibody. Localization of the membranes within the gradient was determined by monitoring chlorophyll absorption (shown on top of the western blot in a,b). (d–f) Liposomes mixed with IM30 (e,f) as well as solely IM30 (d) were separated on a 5–50% SD gradient prior to analysis. The fluorescent probe C6-NBD-PE was incorporated into the liposomes (1:500) for detection, and the position of the liposomes within the gradient was determined by fluorescence spectroscopy. The localization of the liposomes is shown for MGDG/DGDG-containing liposomes and thereafter only indicated above each western blot. The analyses were repeated three times and representative western blots are shown. Full scans of western blots are shown in Supplementary Fig. 8. (g) Peripheral binding of IM30 to liposomes was additionally followed by monitoring changes in the Laurdan fluorescence emission after excitation at 350 nm. Normalized fluorescence emission spectra are shown in the absence (black) and presence of 2.5 μM IM30 (red). Decreased fluorescence emission at 490 nm, that is, increased lipid acyl chain order, indicates peripheral binding of IM30. The experiment was performed four times. See also Supplementary Fig. 2.