Figure 4: Scramblase activity of RP mutants.

(a) Schematic representation of the scramblase activity assay. Details may be found in the text and Supplementary Note 1. (b) Fluorescence traces obtained on adding dithionite to NBD-PC-containing protein-free liposomes (grey trace) and proteoliposomes reconstituted with different opsins (coloured traces) at a high PPR (∼1 g mol⁻¹). Each trace represents the mean of three independent experiments; the variation between experiments was negligible and is contained within the thickness of the lines. (c) Scramblase activity as a function of the amount of WT opsin reconstituted. Scramblase assays were performed for 400 s with vesicles reconstituted at different PPRs (PPR*, in units of grams of protein per mole of phospholipid, obtained by scaling the measured PPR as described in ‘Methods’). The plot represents the dependency of p(≥1) scramblase (the probability of a vesicle having at least one scramblase) on the PPR*. The line represents the data fit calculated as described in ‘Methods’ and in Supplementary Notes 2 and 3. The data are from five independent protein preparations. (d) As in c, for the F45L mutant. The trace for the WT sample is from c. (e) As in d, for the V209M mutant. (f) As in d, for the F220C mutant. Data shown in d–f are from three independent protein preparations in each case. (g) Molar mass (M) of the functionally reconstituted scramblase (mean±s.e., corresponding to Supplementary Table 2) derived from fits of the p(≥1) scramblase versus PPR* data for WT opsin, Ops* and the RP-associated mutants. Dashed lines indicate the molecular weights of opsin monomer and dimer. (h) Schematic illustration of reconstitution modes of WT and RP mutant opsins. All opsins are purified in DDM as monomers. Opsin-DDM micelles are combined with DDM-destabilized vesicles and detergent withdrawal is initiated by adding SM2 BioBeads. When detergent is withdrawn WT opsin dimerizes (or multimerizes), while still within DDM micelles. As DDM continues to be removed from the system, the dimers insert into the available vesicles. For opsin RP mutants, dimerization does not occur and the proteins enter vesicles directly, as monomers.