title: Sterol binding assay using surface plasmon fluorescence spectroscopy.
abstract: We describe the design of a novel in vitro assay to study the interaction of soluble proteins with small hydrophobic sterol ligands. The sterol molecules are incorporated in an artificial membrane system in order to mimic their arrangement found in a biomembrane. The artificial membrane setup is monitored in real time by surface plasmon spectroscopy. Binding of fluorescently labeled soluble protein is observed by optical detection with surface plasmon enhanced fluorescence spectroscopy. By application of the novel assay, we demonstrate that four different oxidized sterol molecules are specifically recognized by the yeast protein Osh5p, a presumed oxysterol binding protein. Osh5p from yeast is the first oxysterol binding protein homologue for which oxysterol binding is shown with this new technique. With the design of our novel in vitro oxysterol binding assay, we have solved the technically challenging difficulty of presenting hydrophobic ligands to hydrophilic proteins in aqueous media.
authors.affiliation: Department of Membrane Biochemistry, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany. wiltschi@biochem.mpg.de
authors.forename: Birgit
authors.initials: B
authors.lastname: Wiltschi
authors.forename: Michael
authors.initials: M
authors.lastname: Schober
authors.forename: Sepp D
authors.initials: SD
authors.lastname: Kohlwein
authors.forename: Dieter
authors.initials: D
authors.lastname: Oesterhelt
authors.forename: Eva-Kathrin
authors.initials: EK
authors.lastname: Sinner
mesh_terms: D004875:Ergosterol / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D008567:Membranes, Artificial; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013261:Sterols / Q000032:analysis / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D000098825:Oxysterol Binding Proteins
chemical_list: D004875:Ergosterol; D008567:Membranes, Artificial; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols; C087902:HES1 protein, S cerevisiae; D000098825:Oxysterol Binding Proteins; C407204:ergosterol epidioxide; C010905:dehydroergosterol