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. 2021 Jun 29;19(6):e3001314. doi: 10.1371/journal.pbio.3001314

Fig 4. Binding of FisB ECD to acidic lipids.

Fig 4

(A) Domain structure of FisB and its His6-tagged ECD used in floatation experiments. (B) Predicted model of FisB44-225 comprising most of the ECD [49], schematically attached to the membrane. (C) Schematic of the floatation assay. Liposomes (40 nmol total lipid) and FisB ECD (200 pmol) were incubated for 1 hour (total volume of 100 μl) at room temperature and layered at the bottom of an iodixanol density gradient. Upon ultracentrifugation, liposomes float to the top interface, whereas unbound protein remains at the bottom. Four fractions were collected as indicated and analyzed by SDS-PAGE. (D) SYPRO orange stained gel of FisB ECD incubated with liposomes containing 45 mole % CL. The percentage of recovered protein is determined by comparing the intensity of the band in fraction B to the input band intensity. (E) Indistinguishable amounts of FisB ECD are recovered when FisB ECD is incubated with liposomes containing different acidic lipid species as long as the charge density is similar. CL30, PG60, and PS60 indicate liposomes containing 30 mole % CL, 60 mole % PG, and 60 mole % PS, respectively. CL carries 2 negative charges, whereas PG and PS carry one each. The rest of the liposome composition is PC. (F) Fraction of liposome-bound iFluor555-FisB ECD (100 nM) recovered after floatation as a function of lipid concentration. Titration curves were fit to fb = K[L]/(1+K[L]), where fb is the bound fraction of protein, [L] is the total lipid concentration (assumed to be ≫ [protein bound]) and K = 1/Kd the apparent association constant, and Kd is the apparent dissociation constant. (G) Best fit values for Kd were 1.0 μM for CL (95% confidence interval, CI = 0.7–2.1 μM) and 3.6 μM for PG (CI = 2.8–5.0 μM), respectively. iFluor555-FisB ECD (100 nM) was incubated with10−8 to 10−4 M lipids for 1 hour at room temperature before flotation. Liposomes contained 45 mole % of CL or PG and 55% PC. CL, cardiolipin; ECD, extracytoplasmic domain; FisB, fission protein B; iFluor555-FisB ECD, iFluor555-labeled FisB ECD; PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine.