Stability of CB₂ in lipid bilayers.

Abstract

<p>A, Temperature-induced unfolding of CB₂ in detergent micelles and lipid bilayers. For stability studies in micelles the purified CB₂-130 in TD buffer supplemented with 10 µM CP-55,940 was subjected to a temperature gradient from 4°C to 74°C at a rate of 1°C/min, 10 µg protein samples withdrawn at indicated time points, mixed with 100 µg lipids POPC/POPS (4∶1 w/w) in 1% CHAPS and diluted 110-fold into cold 10 mM MOPS buffer under vigorous stirring. The activity of CB₂ was analyzed by measuring the G protein activation rates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046290#s4" target="_blank">Materials and Methods</a>. For measurement of thermostability in lipid bilayers either CB₂-proteoliposomes or membrane preparations harboring fusion CB₂-130 were suspended in 10 mM MOPS buffer at a concentration of CB₂ 0.5 ng/µL, subjected to treatment with linear temperature gradient, and analyzed by G protein activation assay. Dotted line depicts the temperature gradient profile. Figure depicts data ± S.D (error bars) of duplicate measurements from representative experiments (n = 3). B, Temperature stability of CB₂ in proteoliposomes and <i>E. coli</i> membranes. Either purified CB₂ receptor reconstituted into POPC:POPS:CHS bilayers or fusion CB₂-130 in <i>E. coli</i> membranes was incubated for 30 min at the temperatures indicated, and the G protein activation assay performed. 4 ng of CB₂ was used in every reaction and measurements were performed upon addition of 2 µM of CP-55,940 to all samples. Data ± S.D. (error bars) of duplicate measurements from representative experiments (n = 3) are presented.</p