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. 2011 May 13;6(5):e19677. doi: 10.1371/journal.pone.0019677

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Galián et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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BmrC/BmrD were reconstituted at lipid to protein ratio of 40 (w/w) by detergent removal from different solubilized mixtures of DDM/lipid/protein. (a) ATPase activity of proteoliposomes was assessed at 4 mM ATP. E. coli: total extract of E. coli phospholipids; PC: egg-phosphatidylcholine; PA: egg-phosphatidic acid; chol: cholesterol; DOPC: dioleoylphosphatidylcholine; DOPG: dioleoylphosphatidylglycerol; CL: cardiolipin. Lipid composition was as follows (w/w): PC/PA 9∶1; PC/PA/chol 7∶1∶2; DOPC/DOPG 9∶1; PC/CL 8∶2. (b) Cryo-electron microscopy of reconstituted proteoliposomes with EPC/EPA. All proteoliposomes were unilamellar and showed a continuous lipid bilayer. Scale bar: 50 nm. (c) ATPase activity measured after reconstitution of BmrC/BmrD with EPC/EPA or E. coli lipids at lipid to protein ratio of 40 (w/w) and 100 (w/w). (d) Protein incorporation in proteoliposomes after fractionation in a sucrose gradient. The amount of BmrC/BmrD in every fraction was measured by quantitation of the protein bands revealed by immunoblot (the intensity of both bands were added) and normalization to the total amount of BmrC/BmrD loaded onto the sucrose gradient (cf. Materials and Methods). Most proteins were reconstituted in proteoliposomes running at 2.5%–5% sucrose without significant aggregates at 30% sucrose.