title: The rhodopsin-transducin complex houses two distinct rhodopsin molecules.
abstract: Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho(∗), which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1mM n-dodecyl-β-d-maltoside (DDM) and Rho monomers in 5mM DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho(∗)·Gt and sConA-Rho(∗)·Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3 D envelope calculated from projections of negatively stained Rho(∗)·Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho(∗)·Gt complexes unequivocally demonstrated a pentameric assembly of the Rho(∗)·Gt complex in which the photoactivated Rho(∗) dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho(∗) dimer in the heteropentameric complex exhibited different capabilities for regeneration with either 11-cis or 9-cis-retinal.
authors: Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4965, USA. bxj27@case.edu
authors: Beata
authors: B
authors: Jastrzebska
authors: Philippe
authors: P
authors: Ringler
authors: Krzysztof
authors: K
authors: Palczewski
authors: Andreas
authors: A
authors: Engel
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003208:Concanavalin A; D019281:Dimerization; D005960:Glucosides; D015151:Immunoblotting; D046911:Macromolecular Substances / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry*; D015293:Transducin / Q000737:chemistry*
chemical_list: D005960:Glucosides; D046911:Macromolecular Substances; D003208:Concanavalin A; C019558:succinylconcanavalin A; C040358:dodecyl maltoside; D012243:Rhodopsin; D015293:Transducin