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 t...

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Veröffentlicht in:Biochemistry (Easton) 2005-07, Vol.44 (29), p.9936-9943
Hauptverfasser: Morizumi, Takefumi, Imai, Hiroo, Shichida, Yoshinori
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Sprache:eng
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Zusammenfassung: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 α-subunit of Gt.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi0504512