title: Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.
abstract: Scavenger receptor class B type I (SR-BI), the high density lipoprotein (HDL) receptor, is important for the delivery of HDL-cholesteryl esters to the liver for excretion via bile formation. The focus on therapeutic strategies aimed at reducing cholesterol levels highlights the critical need to understand the structural features of SR-BI that drive cholesterol removal. Yet, in the absence of a high-resolution structure of SR-BI, our understanding of how SR-BI interacts with HDL is limited. In this study, we have optimized the NMR solution conditions for the structural analysis of the C-terminal transmembrane domain of SR-BI that harbors putative domains required for receptor oligomerization. An isotopically-labeled SR-BI peptide encompassing residues 405-475 was bacterially-expressed and purified. [U-(15)N]-SR-BI(405-475) was incorporated into different detergent micelles and assessed by (1)H-(15)N-HSQC in order to determine which detergent micelle best maintained SR-BI(405-475) in a folded, native conformation for subsequent NMR analyses. We also determined the optimal detergent concentration used in micelles, as well as temperature, solution buffer and pH conditions. Based on (1)H-(15)N-HSQC peak dispersion, intensity, and uniformity, we determined that [U-(15)N]-SR-BI(405-475) should be incorporated into 5% detergent micelles consisting of 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-[1'-rac-glycerol] (LPPG) and data collected at 40°C in a non-buffered solution at pH 6.8. Furthermore, we demonstrate the ability of SR-BI(405-475) to form dimers upon chemical crosslinking. These studies represent the first steps in obtaining high-resolution structural information by NMR for the HDL receptor that plays a critical role in regulating whole body cholesterol removal.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Chadwick
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Davin R
authors.initials: DR
authors.lastname: Jensen
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Francis C
authors.initials: FC
authors.lastname: Peterson
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. Electronic address: bvolkman@mcw.edu.
authors.forename: Brian F
authors.initials: BF
authors.lastname: Volkman
authors.affiliation: Department of Medicine, Division of Endocrinology, Metabolism & Clinical Nutrition, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA; Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA; Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. Electronic address: dsahoo@mcw.edu.
authors.forename: Daisy
authors.initials: D
authors.lastname: Sahoo
mesh_terms: D000818:Animals; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008823:Micelles; D017434:Protein Structure, Tertiary; D051122:Scavenger Receptors, Class B / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D003902:Detergents; D008823:Micelles; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B
keywords: Detergent; Expression; HDL; Micelles; NMR; Purification; SR-BI; Transmembrane