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Purification of the Rhodopsin–Transducin Complex for Structural Studies
- Source :
- Methods in Molecular Biology ISBN: 9781493995318
- Publication Year :
- 2019
- Publisher :
- Springer New York, 2019.
-
Abstract
- G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and are targets for over 30% of all drugs on the market. Structural information of GPCRs and more importantly that of the complex between GPCRs and their signaling partner heterotrimeric G proteins is of great importance. Here we present a method for the large-scale purification of the rhodopsin-transducin complex, the GPCR-G protein signaling complex in visual phototransduction, directly from their native retinal membrane using native proteins purified from bovine retinae. Formation of the complex on native membrane is orchestrated in part by the proper engagement of lipid-modified rhodopsin and transducin (i.e., palmitoylation of the rhodopsin C-terminus, myristoylation and farnesylation of the αT and γ1, respectively). The resulting complex is of high purity and stability and has proved suitable for further biophysical and structural studies. The methods described here should be applicable to other recombinantly expressed receptors from insect cells or mamalian cells by forming stable, functional complexes directly on purified cell membranes.
- Subjects :
- 0301 basic medicine
genetic structures
biology
G protein
Chemistry
05 social sciences
Cell biology
03 medical and health sciences
030104 developmental biology
Palmitoylation
Rhodopsin
Heterotrimeric G protein
0502 economics and business
biology.protein
050211 marketing
sense organs
Transducin
Visual phototransduction
Myristoylation
G protein-coupled receptor
Subjects
Details
- ISBN :
- 978-1-4939-9531-8
- ISBNs :
- 9781493995318
- Database :
- OpenAIRE
- Journal :
- Methods in Molecular Biology ISBN: 9781493995318
- Accession number :
- edsair.doi...........05c2712c73567238e48a21d9181b6944