Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist

Biochemistry. 2007 Oct 30;46(43):12248-52. doi: 10.1021/bi701003b. Epub 2007 Oct 6.

Abstract

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Cattle
  • Chlorocebus aethiops
  • Models, Molecular
  • Mutation
  • Protein Engineering*
  • Rhodopsin / agonists
  • Rhodopsin / antagonists & inhibitors
  • Rhodopsin / chemistry*
  • Rhodopsin / genetics
  • Rhodopsin / isolation & purification
  • Spectrophotometry, Ultraviolet

Substances

  • Rhodopsin