Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis. Protein Expr Purif 2015 Mar;107:35-42 PMID: 25461971 PMCID: PMC4270826

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.

Author List

Chadwick AC, Jensen DR, Peterson FC, Volkman BF, Sahoo D

Authors

Francis C. Peterson PhD Professor in the Biochemistry department at Medical College of Wisconsin
Daisy Sahoo PhD Vice Chair, Associate Professor in the Medicine department at Medical College of Wisconsin
Brian F. Volkman PhD Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Detergents
Dimerization
Escherichia coli
Gene Expression
Magnetic Resonance Spectroscopy
Mice
Micelles
Protein Structure, Tertiary
Scavenger Receptors, Class B



View this publication's entry at the Pubmed website PMID: 25461971
jenkins-FCD Prod-158 788e756cee95f97989f23472575fc754037544be