title: Dynamics of arrestin-rhodopsin interactions: loop movement is involved in arrestin activation and receptor binding.
abstract: In this study we investigate conformational changes in Loop V-VI of visual arrestin during binding to light-activated, phosphorylated rhodopsin (Rho*-P) using a combination of site-specific cysteine mutagenesis and intramolecular fluorescence quenching. Introduction of cysteines at positions in the N-domain at residues predicted to be in close proximity to Ile-72 in Loop V-VI of arrestin (i.e. Glu-148 and Lys-298) appear to form an intramolecular disulfide bond with I72C, significantly diminishing the binding of arrestin to Rho*-P. Using a fluorescence approach, we show that the steady-state emission from a monobromobimane fluorophore in Loop V-VI is quenched by tryptophan residues placed at 148 or 298. This quenching is relieved upon binding of arrestin to Rho*-P. These results suggest that arrestin Loop V-VI moves during binding to Rho*-P and that conformational flexibility of this loop is essential for arrestin to adopt a high affinity binding state.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239-3098, USA.
authors.forename: Martha E
authors.initials: ME
authors.lastname: Sommer
authors.forename: David L
authors.initials: DL
authors.lastname: Farrens
authors.forename: J Hugh
authors.initials: JH
authors.lastname: McDowell
authors.forename: Lauren A
authors.initials: LA
authors.lastname: Weber
authors.forename: W Clay
authors.initials: WC
authors.lastname: Smith
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019393:Arrestin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D019393:Arrestin; D001643:Bridged Bicyclo Compounds; D012243:Rhodopsin; D003545:Cysteine; C028381:monobromobimane