Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin

Jastrzebska, Beata; Golczak, Marcin; Fotiadis, Dimitrios; Engel, Andreas; Palczewski, Krzysztof (2009). Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin. FASEB journal, 23(2), pp. 371-81. Bethesda, Md.: Federation of American Societies for Experimental Biology 10.1096/fj.07-114835

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Transitory binding between photoactivated rhodopsin (Rho* or Meta II) and the G protein transducin (Gt-GDP) is the first step in the visual signaling cascade. Light causes photoisomerization of the 11-cis-retinylidene chromophore in rhodopsin (Rho) to all-trans-retinylidene, which induces conformational changes that allow Gt-GDP to dock onto the Rho* surface. GDP then dissociates from Gt, leaving a transient nucleotide-empty Rho*-Gt(e) complex before GTP becomes bound, and Gt-GTP then dissociates from Rho*. Further biochemical advances are required before structural studies of the various Rho*-Gt complexes can be initiated. Here, we describe the isolation of n-dodecyl-beta-maltoside solubilized, stable, functionally active, Rho*-Gt(e), Rho(e)*-Gt(e), and 9-cis-retinal/11-cis-retinal regenerated Rho-Gt(e) complexes by sucrose gradient centrifugation. In these complexes, Rho* spectrally remained in its Meta II state, and Gt(e) retained its ability to interact with GTPgammaS. Removal of all-trans-retinylidene from Rho*-Gt(e) had no effect on the stability of the Rho(e)*-Gt(e) complex. Moreover, opsin in the Rho(e)*-Gt(e) complex with an empty nucleotide-binding pocket in Gt and an empty retinoid-binding pocket in Rho was regenerated up to 75% without complex dissociation. These results indicate that once Rho* couples with Gt, the chromophore plays a minor role in stabilizing this complex. Moreover, in complexes regenerated with 9-cis-retinal/11-cis-retinal, Rho retains a conformation similar to Rho* that is stabilized by Gt(e) apo-protein.

Item Type:	Journal Article (Original Article)
Division/Institute:	04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine
UniBE Contributor:	Fotiadis, Dimitrios José
ISSN:	0892-6638
Publisher:	Federation of American Societies for Experimental Biology
Language:	English
Submitter:	Factscience Import
Date Deposited:	04 Oct 2013 15:12
Last Modified:	05 Dec 2022 14:22
Publisher DOI:	10.1096/fj.07-114835
PubMed ID:	18827025
Web of Science ID:	000262892900009
URI:	https://boris.unibe.ch/id/eprint/31629 (FactScience: 196260)