title: Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy.
abstract: The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype G protein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε-(15)N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The meta II and meta III states emerge in parallel with a relative ratio of about 3:1. Transient formation of the meta III state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2 -loop in the formation of the meta III state.
authors: Institute for Organic Chemistry and Chemical Biology, Center of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany).
authors: Jochen
authors: J
authors: Stehle
authors: Robert
authors: R
authors: Silvers
authors: Karla
authors: K
authors: Werner
authors: Deep
authors: D
authors: Chatterjee
authors: Santosh
authors: S
authors: Gande
authors: Frank
authors: F
authors: Scholz
authors: Arpana
authors: A
authors: Dutta
authors: Josef
authors: J
authors: Wachtveitl
authors: Judith
authors: J
authors: Klein-Seetharaman
authors: Harald
authors: H
authors: Schwalbe
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D057809:HEK293 Cells; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes / Q000737:chemistry; D010786:Photoreceptor Cells / Q000378:metabolism; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012996:Solutions / Q000737:chemistry
chemical_list: D009587:Nitrogen Isotopes; D012996:Solutions; C013110:metarhodopsins; D012243:Rhodopsin
keywords: G-protein coupled receptors; NMR spectroscopy; bovine rhodopsin; membrane protein dynamics; photocycles