Direct observation of the complex formation of GDP-bound transducin with the rhodopsin intermediate having a visible absorption maximum in rod outer segment membranes.

Rhodopsin is a photoreceptive protein that is present in rod photoreceptor cells, inducing a GDP-GTP exchange reaction on the retinal G-protein transducin (Gt) upon light absorption. This exchange reaction proceeds through at least three steps, which include the binding of photoactivated rhodopsin to GDP-bound Gt, the dissociation of GDP from the rhodopsin-Gt complex, and the binding of GTP to the nucleotide-unbound Gt. These steps have been thought to occur after the formation of the rhodopsin intermediate, meta-II; however, the extra formation of meta-II, which reflects the formation of a complex with Gt, was inhibited in the presence of excess GDP. Here, we use a newly developed CCD spectrophotometer to show that a meta-II precursor, meta-Ib, which has an absorption maximum at visible region, can bind to Gt in its GDP-bound form in urea-washed bovine rod outer segment membranes. The affinity of meta-Ib for GDP-bound Gt is about two times less than that of meta-II for GDP-unbound Gt, indicating that the extra formation of meta-II is observed at equilibrium even in the presence of the meta-Ib-Gt complex. This is the first identification of a complex that includes the GDP-bound form of G protein. Our results strongly suggest that the protein conformational change of the rhodopsin intermediate after binding to Gt is important for the induction of the nucleotide release from the alpha-subunit of Gt.