title: Conformational equilibria of light-activated rhodopsin in nanodiscs.
abstract: Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095.|Jules Stein Eye Institute, University of California, Los Angeles, CA 90095.
authors.forename: Ned
authors.initials: N
authors.lastname: Van Eps
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: Lydia N
authors.initials: LN
authors.lastname: Caro
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: Takefumi
authors.initials: T
authors.lastname: Morizumi
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095.|Jules Stein Eye Institute, University of California, Los Angeles, CA 90095.
authors.forename: Ana Karin
authors.initials: AK
authors.lastname: Kusnetzow
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany.
authors.forename: Michal
authors.initials: M
authors.lastname: Szczepek
authors.affiliation: Institut für Medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany.
authors.forename: Klaus Peter
authors.initials: KP
authors.lastname: Hofmann
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Champaign, IL 61801.
authors.forename: Timothy H
authors.initials: TH
authors.lastname: Bayburt
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Champaign, IL 61801.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; oliver.ernst@utoronto.ca hubbellw@jsei.ucla.edu.|Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: Oliver P
authors.initials: OP
authors.lastname: Ernst
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; oliver.ernst@utoronto.ca hubbellw@jsei.ucla.edu.|Jules Stein Eye Institute, University of California, Los Angeles, CA 90095.
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D000818:Animals; D002417:Cattle; D008027:Light; D049329:Nanostructures / Q000737:chemistry*; D011487:Protein Conformation / Q000528:radiation effects*; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D013113:Spin Labels; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects
chemical_list: D013113:Spin Labels; D012243:Rhodopsin; D015293:Transducin
keywords: GPCR; conformational heterogeneity; double electron–electron resonance; nanodiscs; rhodopsin