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_T). This results in the dissociation of G_T into its component α_T-GTP and β₁γ₁ subunit complex. Structural information for the Rho*-G_T 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_T*) comprising a Gα_T/Gα_i1 chimera (α_T*) and β₁γ₁ 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_T* 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_T*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β₂-adrenergic receptor-G_S complex, including a flexible α_T* 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.

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