Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.

The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (G). This results in the dissociation of G into its component α-GTP and βγ subunit complex. Structural information for the Rho*-G complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (G*) comprising a Gα/Gα chimera (α*) and βγ The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to Gα* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one G*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β-adrenergic receptor-G complex, including a flexible α* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.