Figure 5. Scramblase activity assay of HflK/C^SS in the presence or absence of DTT, the M1 mutant, and wild-type HflK/C.

(A) Schematic representation of the scramblase activity assay (partially created with BioRender.com). In a protein-free liposome (top), addition of dithionite quenches only NBD fluorophores in the outer leaflet, producing ~50% fluorescence loss. In the presence of an active scramblase (bottom), NBD-labeled lipids are translocated between leaflets, continuously exposing inner-leaflet NBD-labeled lipids to the outer leaflet, where they are quenched by dithionite, resulting in >50% fluorescence loss.

(B) HflK/C^SS in the closed (−DTT) and open (+DTT) conformations, as well as the M1 mutant, shows greater than 50% fluorescence reduction, indicating lipid scramblase activity similar to that of wild-type HflK/C, whereas liposomes alone show only ~50% quenching. All assays were performed using a 50 nM complex (as determined by a BSA assay through quantification of the HflK band) at 30°C. Values represent means (n = 3 technical replicates), with error bars indicating the SD.