Abstract
Transitory binding between photoactivated rhodopsin (Rho* or Meta II) and the G protein transducin (Gt-GDP) is the first step in the visual signaling cascade. Light causes photoisomerization of the 11-cis-retinylidene chromophore in rhodopsin (Rho) to all-trans-retinylidene, which induces conformational changes that allow Gt-GDP to dock onto the Rho* surface. GDP then dissociates from Gt, leaving a transient nucleotide-empty Rho*-Gt(e) complex before GTP becomes bound, and Gt-GTP then dissociates from Rho*. Further biochemical advances are required before structural studies of the various Rho*-Gt complexes can be initiated. Here, we describe the isolation of n-dodecyl-beta-maltoside solubilized, stable, functionally active, Rho*-Gt(e), Rho(e)*-Gt(e), and 9-cis-retinal/11-cis-retinal regenerated Rho-Gt(e) complexes by sucrose gradient centrifugation. In these complexes, Rho* spectrally remained in its Meta II state, and Gt(e) retained its ability to interact with GTPgammaS. Removal of all-trans-retinylidene from Rho*-Gt(e) had no effect on the stability of the Rho(e)*-Gt(e) complex. Moreover, opsin in the Rho(e)*-Gt(e) complex with an empty nucleotide-binding pocket in Gt and an empty retinoid-binding pocket in Rho was regenerated up to 75% without complex dissociation. These results indicate that once Rho* couples with Gt, the chromophore plays a minor role in stabilizing this complex. Moreover, in complexes regenerated with 9-cis-retinal/11-cis-retinal, Rho retains a conformation similar to Rho* that is stabilized by Gt(e) apo-protein.