Journal
ANALYTICAL BIOCHEMISTRY
Volume 323, Issue 1, Pages 139-149Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2003.08.029
Keywords
mass spectrometry; electrospray ionization; noncovalent complex; retinoic acid receptor-related orphan receptor alpha; ROR alpha; cholesterol
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The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORalpha-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization-mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography-mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D-3; 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORalpha-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules. (C) 2003 Elsevier Inc. All rights reserved.
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