The rhodopsin-transducin complex houses two distinct rhodopsin molecules

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• 作者：Beata Jastrzebska^a ; ^{bxj27@case.edu} ; Philippe Ringler^b ; Krzysztof Palczewski^a ; ^{kxp65@case.edu} ; Andreas Engel^a ; ^b ; ^{andreas.engel@case.edu} ; ^{andreas.engel@unibas.ch}
• 关键词：b2AR ; ¦Â2-adrenergic receptor ; Bis&ndash ; Tris propane ; 1 ; 3-bis[tris(hydroxymethyl)-methylamino]propane ; CTF ; contrast transfer function ; DDM ; n-dodecyl-¦Â-d-maltoside ; DSG ; disuccinimidyl glutarate ; DTT ; dithiothreitol ; GDP ; guanosine diphosphate ; GTP ; gu
• 刊名：Journal of Structural Biology
• 出版年：2013
• 出版时间：May, 2013
• 年：2013
• 卷：182
• 期：2
• 页码：164-172
• 全文大小：2176 K

文摘

Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho^?, which binds the heterotrimeric G protein, transducin (G_t) causing GDP to GTP exchange and G_t dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1 mM n-dodecyl-¦Â-d-maltoside (DDM) and Rho monomers in 5 mM DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho^?¡¤G_t and sConA-Rho^?¡¤G_t complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3 D envelope calculated from projections of negatively stained Rho^?¡¤G_t-LMNG complexes accommodated two Rho molecules, one G_t heterotrimer and a detergent belt. Visualization of triple sConA-Rho^?¡¤G_t complexes unequivocally demonstrated a pentameric assembly of the Rho^?¡¤G_t complex in which the photoactivated Rho^? dimer serves as a platform for binding the G_t heterotrimer. Importantly, individual monomers of the Rho^? dimer in the heteropentameric complex exhibited different capabilities for regeneration with either 11-cis or 9-cis-retinal.