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.
Morizumi
Takefumi
T
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan, and CREST, Japan Science and Technology Agency, Japan.
Imai
Hiroo
H
Shichida
Yoshinori
Y
D000042:Absorption; D000818:Animals; D002417:Cattle; D006153:Guanosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D011485:Protein Binding; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods; D013696:Temperature; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism
D006153:Guanosine Diphosphate; D012243:Rhodopsin; D015293:Transducin