Abstract
We prepared rhodopsin mutants that contained a single reactive cysteine residue per rhodopsin molecule at position 65, 140, 240, or 316 on the cytoplasmic face. A carbene-generating photoactivatable group was linked by a disulfide bond to the cysteine sulfhydryl group of each of the rhodopsin mutants. The resulting derivative was then light-activated at λ > 495 nm to form the metarhodopsin II intermediate, which bound transducin. Subsequent photoactivation (355 nm) of the carbene-generating group resulted in crosslinking of the rhodopsin mutant carrying a cysteine residue at position 240 to transducin. This crosslinking was determined to be specifically with the α subunit of transducin. An alternative reaction observed during photolysis of the rhodopsin mutants was intramolecular insertion of the carbene into rhodopsin.
Original language | English (US) |
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Pages (from-to) | 7643-7647 |
Number of pages | 5 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 91 |
Issue number | 16 |
DOIs | |
State | Published - Aug 2 1994 |
Externally published | Yes |
Keywords
- GTP-binding protein
- phenyldiazirine
- photoactivatable group
- signal transduction
- site-specific mutagenesis
ASJC Scopus subject areas
- General