Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin

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‐β‐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 GTPγS. 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.—Jastrzebska, B., Golczak, M., Fotiadis, D., Engel, A., and Palczewski, K.. Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin. FASEB J . 23, 371–381 (2009)