title: Preparation of an activated rhodopsin/transducin complex using a constitutively active mutant of rhodopsin.
abstract: The interaction of rhodopsin and transducin has been the focus of study for more than 30 years, but only recently have efforts to purify an activated complex in detergent solution materialized. These efforts have used native rhodopsin isolated from bovine retina and employed either sucrose density gradient centrifugation or size exclusion chromatography to purify the complex. While there is general agreement on most properties of the activated complex, subunit stoichiometry is not yet settled, with rhodopsin/transducin molar ratios of both 2/1 and 1/1 reported. In this report, we introduce methods for preparation of the complex that include use of recombinant rhodopsin, so as to take advantage of mutations that confer constitutive activity and enhanced thermal stability on the protein, and immunoaffinity chromatography for purification of the complex. We show that chromatography on ConA-Sepharose can substitute for the immunoaffinity column and that bicelles can be used instead of detergent solution. We demonstrate the following: that rhodopsin has a covalently bound all-trans-retinal chromophore and therefore corresponds to the active metarhodopin II state; that transducin has an empty nucleotide-binding pocket; that the isolated complex is active and dissociates upon addition of guanine nucleotide; and finally that the stoichiometry corresponds reproducibly to a 1/1 molar ratio of rhodopsin to transducin.
authors: Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, United States.
authors: Guifu
authors: G
authors: Xie
authors: Aaron M
authors: AM
authors: D'Antona
authors: Patricia C
authors: PC
authors: Edwards
authors: Maikel
authors: M
authors: Fransen
authors: Jorg
authors: J
authors: Standfuss
authors: Gebhard F X
authors: GF
authors: Schertler
authors: Daniel D
authors: DD
authors: Oprian
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000379:methods*; D000818:Animals; D002417:Cattle; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D012160:Retina / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015293:Transducin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D012243:Rhodopsin; D015293:Transducin