Figure 7.

Analysis of phospholipids in the local lipid polymer-nanodiscs. (A) TLC of lipids present in membranes and nanodiscs was identified by running pure synthetic samples of each as a standard (not shown). Bands above the labeled lipids are attributed to photoreaction center ([RC] from the purple bacterium Rhodobacter sphaeroides) pigments. DDM was visualized, but LDAO did not stain. Additional bands in the DDM and LDAO profiles are unidentified. No lipids could be detected in the samples solubilized by detergents DDM and LDAO, indicating the detergent removal of lipids [133]. (B) TLC of chloroform-methanol extracts of the yeast mitochondria and of CytcO-SMA native nanodiscs. From left to right: the 1st and the 2nd lanes are lipid standards (0.04 mg CL, 0.05 mg of each of DOPC, DOPE, PI, and PS); the 3rd lane is the extract of the yeast mitochondria (the loaded lipids were extracted from a sample that originally contained ~0.6 mg protein); the 4th and 5th lanes are extracts of two preparations of CytcO-SMA. The loaded lipids (at the arrow) were extracted from a sample that originally contained ~0.2 mg protein. The bands in lane 4 are slightly weaker than those in lane 5, presumably because slightly less material was applied in the former. The bands were visualized by iodine staining [113]. (C) Mitochondria subjected to mock SMA incubation (lane 4), or incubated with SMA, to form mitochondrial-LipodisqsW (lane 5) were analyzed for lipid content by thin-layer chromatography. Lipid standards are shown in lanes 1–3 [130]. (D) ³¹P NMR spectra of native E. coli lipids present in the polymer-nanodiscs and of the synthetic lipids (a reference sample). PE: phosphatidylethanolamine, CL: cardiolipin, and PG: phosphatidylglycerol, * indicates the uncharacterized E. coli lipids [55]. (E,F) Mass spectrometry analysis of SMA extracted AcrB after exchange into A8–35 and DDM. Initial native MS results of A8–35_Ex (E) and DDM_Ex (F) [134]. Figure 7A,B and captions are adopted from the references [113,133] with copyright permission. Figure 7C–F and captions are adopted from the references [55,130,134].