Structuring Detergents for Extracting and Stabilizing Functional Membrane Proteins
Figure 5
C4Cn preserve a functional state of BmrA.
(A) Binding of daunorubicin to BmrA monitored by intrinsic (Tryptophan, Trp) fluorescence quenching. BmrA was extracted and purified either with C4C10 (circles) or FC12 (triangles) as in Figure 4, the former being subsequently exchanged by FC12 as in Figure 4D. The purified protein was then incubated with increasing concentrations of daunorubicin, the binding of the drug being probed by the variation of intrinsic fluorescence of BmrA, as described in Methods. (B) VO4-sensitive ATPase activity of BmrA (see Methods) in different fractions: BmrA-enriched membrane fraction (“-“ bar) corresponding to 0.5 µmol/min.mg and taken as 100%; BmrA-enriched membrane fraction solubilized with 1% SDS, FC12, DDM, or C4C3+C4C7 (“Extraction/C4Cn” bar, carried out as in Fig. 4); BmrA extracted with FC12 and then purified by metal affinity and gel filtration with FC12 (“Purification/FC12” bar); BmrA extracted with C4C3+C4C7 and then purified by metal affinity with C4C7 followed by detergent exchange with FC12 using gel filtration as carried out in Figure 4 (“Purification/C4Cn, FC12 exchange” bar). (C) Intrinsic fluorescence quenching monitoring of C4C10 binding on BmrA. BmrA was extracted either with C4C10 or FC12 and then purified with FC12 as in Figure 4 generating two populations on which C4C10 binding was monitored by probing the quenching of intrinsic fluorescence of 1 µM BmrA as detailed in Methods.