The rhodopsin-transducin complex houses two distinct rhodopsin molecules
Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho∗, which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1 mM n-dodecyl-Î²-d-maltoside (DDM) and Rho monomers 5 mM in DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho∗·Gt and sConA-Rho∗·Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3-D envelope calculated from projections of negatively stained Rho∗·Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho∗·Gt complexes unequivocally demonstrated a pentameric assembly of the Rho∗·Gt complex in which the photoactivated Rho∗ dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho∗ dimer in the heteropentameric complex exhibited different capabilities to be regenerated with either 11-cis or 9-cis-retinal.