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

Biochemistry. 2005 Jul 26;44(29):9936-43. doi: 10.1021/bi0504512.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Absorption
  • Animals
  • Cattle
  • Guanosine Diphosphate / chemistry*
  • Guanosine Diphosphate / metabolism
  • Models, Chemical
  • Protein Binding
  • Rhodopsin / chemistry*
  • Rhodopsin / metabolism
  • Rod Cell Outer Segment / chemistry*
  • Rod Cell Outer Segment / metabolism
  • Spectrophotometry / instrumentation
  • Spectrophotometry / methods
  • Temperature
  • Transducin / chemistry*
  • Transducin / metabolism

Substances

  • Guanosine Diphosphate
  • Rhodopsin
  • Transducin