Stabilizing effects of POPC:POPS (1∶1, w/w) in micelles.

Abstract

<p>A, Lipids were added to DDM/CHAPS (0.1%/0.5%) micelles at concentrations indicated. Upon incubation samples were supplemented with POPC:POPS dissolved in 1% CHAPS so that the final protein-to-lipid ratio was the same in all samples (1∶500 mol/mol). Upon reconstitution on a mini-spin detergent-absorbent columns the activity of CB₂ was determined by the G protein activation assay and reported as % of the maximal activity measured for this series of samples. The liposomes-reconstituted receptor (0.1% CHS in micelles, POPC/POPS/CHS 60∶15:25 in liposomes) exhibiting the highest levels of activation in this experiment was used as an activity standard. B, Purified CB₂ in DDM/CHAPS/CHS micelles was captured on Ni-NTA, detergent buffer rapidly exchanged to DDM/CHAPS containing 0.4% of lipids of indicated composition, protein eluted with imidazole and liposome-reconstituted by rapid dilution. Functional activity of CB₂ reconstituted into POPC/POPS/CHS matrix is set as 100% of activity. Figures depict data ± SD (error bars) of duplicate determinations from representative experiments (n = 2-3).</p