Rhodopsin-transducin heteropentamer: Three-dimensional structure and biochemical characterization

The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho∗ form. Rho∗ binds the heterotrimeric G protein, transducin (Gt) inducing GDP to GTP exchange and Gt dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho∗·Gt complex. Quantitative SDS-PAGE suggested a 2:1 molar ratio of Rho∗ to Gt in the complex and its mass determined by scanning transmission electron microscopy was 221 ± 12 kDa. A 21.6 Å structure was calculated from projections of negatively stained Rho∗·Gt complexes. The molecular envelope thus determined accommodated two Rho molecules together with one Gt heterotrimer, corroborating the heteropentameric structure of the Rho∗·Gt complex.