Abstract
Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin α-subunit (GTα) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the GTα. C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the GTα C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the GTα C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.
Original language | English (US) |
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Pages (from-to) | 29767-29773 |
Number of pages | 7 |
Journal | Journal of Biological Chemistry |
Volume | 279 |
Issue number | 28 |
DOIs | |
State | Published - Jul 9 2004 |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology