Improvements in G protein-coupled receptor purification yield light stable rhodopsin crystals.
G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling proteins and are the target of approximately half of all therapeutic agents. Agonist ligands bind their cognate GPCRs stabilizing the active conformation that is competent to bind G proteins, thus initiating a cascade of intracellular signaling events leading to modification of the cell activity. Despite their biomedical importance, the only known GPCR crystal structures are those of inactive rhodopsin forms. In order to understand how GPCRs are able to transduce extracellular signals across the plasma membrane, it is critical to determine the structure of these receptors in their ligand-bound, active state. Here, we report a novel combination of purification procedures that allowed the crystallization of rhodopsin in two new crystal forms and can be applicable to the purification and crystallization of other membrane proteins. Importantly, these new crystals are stable upon photoactivation and the preliminary X-ray diffraction analysis of both photoactivated and ground state rhodopsin crystals are also reported.
Salom
David
D
Novasite Pharmaceuticals, Inc., San Diego, CA 92121, USA. DSalom@Novasite.com
Le Trong
Isolde
I
Pohl
Ehmke
E
Ballesteros
Juan A
JA
Stenkamp
Ronald E
RE
Palczewski
Krzysztof
K
Lodowski
David T
DT
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D015925:Cryopreservation; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D008024:Ligands; D008027:Light; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000302:isolation & purification; D012243:Rhodopsin / Q000737:chemistry* / Q000302:isolation & purification; D013997:Time Factors; D014961:X-Ray Diffraction / Q000379:methods
D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D012243:Rhodopsin; D000645:Ammonium Sulfate