Abstract
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 β(1)γ(1) 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 β(1)γ(1) 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 β(2)-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.