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Design, synthesis and characterization of CHAPSTEROL, a novel choles-terol-based detergent developed for functional solubilization of cholesterol-dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein-coupled receptor requiring cholesterol for its high-affinity binding state. Using the photoactivatable cholesterol analogue [ 3 H]6,6-azocholestan-3b-ol[3aH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol-binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol-binding proteins | iFEBS Journal CHAPSTEROL A novel cholesterol-based detergent Katja Gehrig-Burger1 Ladislav Kohout2 and Gerald Gimpl1 1 Institute of Biochemistry University of Mainz Germany 2 Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague Czech Republic Keywords CHAPSTEROL detergent photoreactive cholesterol rafts steroid nucleus Correspondence K. Gehrig-Burger Institute of Biochemistry University of Mainz Becherweg 30 55099 Mainz Germany Fax 49 6131 3925348 Tel 49 6131 3923829 E-mail kburger@uni-mainz.de Website http www.bio.chemie. uni-mainz.de Gehrig KG_de.htm Received 19 October 2004 revised 5 December 2004 accepted 7 December 2004 Design synthesis and characterization of CHAPSTEROL a novel cholesterol-based detergent developed for functional solubilization of cholesteroldependent membrane proteins are described. To validate CHAPSTEROL we employed the oxytocin receptor a G protein-coupled receptor requiring cholesterol for its high-affinity binding state. Using the photoactivatable cholesterol analogue 3H 6 6-azocholestan-3b-ol 3aH we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol-binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol-binding proteins. Similar to Triton X-100 and CHAPS CHAPSTEROL maintains the localization of caveolin as well as cholesterol and sphingomyelin to lipid rafts i.e. detergent-insoluble microdomains of the plasma membrane. The data suggest that CHAPSTEROL is an appropriate detergent for the solubilization of cholesterol-dependent membrane proteins and isolation of rafts. doi 10.1111 j.1742-4658.2004.04517.x The plasma membrane of eukaryotic cells is a highly organized and dynamic structure. Lipids and proteins are asymmetrically distributed between the leaflets of the membrane bilayer. Several findings also indicate an asymmetrical distribution of lipids between lateral subdomains of the membrane. One .