Assembly of an Activated Rhodopsin–Transducin Complex in Nanoscale Lipid Bilayers

• Published in: Biochemistry (Easton) Vol. 53; no. 1; pp. 127 - 134
• Main Authors: D’Antona, Aaron M, Xie, Guifu, Sligar, Stephen G, Oprian, Daniel D
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 14.01.2014
• Subjects: Guanine Nucleotides - metabolism; Lipid Bilayers - chemistry; Nanostructures; Rhodopsin - chemistry; Rhodopsin - genetics; Rhodopsin - metabolism; Transducin - chemistry; Transducin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi4012995

The formation and characterization of an activated complex of the visual pigment rhodopsin and its downstream signaling partner transducin have been the subject of intense focus by several research groups. While the subunit composition of the activated complex is still the subject of some controversy, our laboratory [Xie, G., D’Antona, A. M., Edwards, P. C., Fransen, M., Standfuss, J., Schertler, G. F. X., and Oprian, D. D. (2011) Biochemistry 50, 10399–10407] and that of Ernst et al. [Ernst, O. P., Gramse, V., Kolbe, M., Hofmann, K. P., and Heck, M. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 10859–10864] find that the two proteins are present in a 1/1 molar ratio. Unfortunately, these data could not distinguish a ratio of 1/1 from ratios of 2/2, 3/3, etc. For this reason, we reinvestigated the issue of stoichiometry of the activated complex, exploiting the ability of Nanodisc lipid bilayers to isolate single molecules of rhodopsin. We show here that the purified complex in Nanodiscs contains an activated rhodopsin with a covalently bound all-trans-retinal chromophore, that transducin has an empty nucleotide-binding pocket, that the isolated complex is active and dissociates upon addition of guanine nucleotide, and that the stoichiometry corresponds to exactly one molecule of rhodopsin and one molecule of transducin.