ArticlePloS one2014
Crystallization scale preparation of a stable GPCR signaling complex between constitutively active rhodopsin and G-protein.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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18 citing papers in PubMed, 27 citations in OpenAlex.
- Probiotics in the Prophylaxis of Premature Rupture of Membranes and Cervical Incompetence.Nutrients · 2024Review
- Active state structures of a bistable visual opsin bound to G proteins.Nature communications · 2024Article
- Why Are We Still Cloning Melatonin Receptors? A Commentary.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Biochemical Characterization of GPCR-G Protein Complex Formation.Methods in molecular biology (Clifton, N.J.) · 2021Article
- Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization.Journal of visualized experiments : JoVE · 2020Article
- Crystal structure of jumping spider rhodopsin-1 as a light sensitive GPCR.Proceedings of the National Academy of Sciences of the United States of America · 2019Article
- Article
- Structural biology of G protein-coupled receptor signaling complexes.Protein science : a publication of the Protein Society · 2019Review
- Coupling of Human Rhodopsin to a Yeast Signaling Pathway Enables Characterization of Mutations Associated with Retinal Disease.Genetics · 2019Article
- In vivo assembly and large-scale purification of a GPCR - Gα fusion with Gβγ, and characterization of the active complex.PloS one · 2019Article
- Development of an antibody fragment that stabilizes GPCR/G-protein complexes.Nature communications · 2018Article
- Crystal structure of rhodopsin in complex with a mini-GScience advances · 2018Article
- Structure of the µ-opioid receptor-GNature · 2018Article
- Article
- Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.The Journal of biological chemistry · 2017Article
- Probing Gαi1 protein activation at single-amino acid resolution.Nature structural & molecular biology · 2015Article
- Stabilization of G protein-coupled receptors by point mutations.Frontiers in pharmacology · 2015Review
- Chemistry and biology of the initial steps in vision: the Friedenwald lecture.Investigative ophthalmology & visual science · 2014Article
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Authors and funding
10 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine retinas. We demonstrate that the light-activated rhodopsin in this complex contains a covalently bound unprotonated retinal and therefore corresponds to the active metarhodopin II state; that the isolated complex is active and dissociates upon addition of GTPγS; and that the stoichiometry corresponds to a 1∶1 molar ratio of rhodopsin to the heterotrimeric G-protein. And finally, we show that the rhodopsin also forms stable complex with Gi. This complex has significantly higher thermostability than RhoM257Y/Gt complex and is resistant to a variety of detergents. Overall, our data suggest that the RhoM257Y/Gi complex is an ideal target for future structural and mechanistic studies of signaling in the visual system.
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