Structure and function in rhodopsin: Covalent crosslinking of the rhodopsin (metarhodopsin II)-transducin complex - The rhodopsin cytoplasmic face links to the transducin α subunit

John F. Resek, David Farrens, H. Gobind Khorana

Research output: Contribution to journalArticle

35 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)7643-7647
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number16
DOIs
StatePublished - Aug 2 1994
Externally publishedYes

Fingerprint

Transducin
Rhodopsin
Cysteine
Photolysis
Disulfides
metarhodopsins
Light
carbene

Keywords

  • GTP-binding protein
  • phenyldiazirine
  • photoactivatable group
  • signal transduction
  • site-specific mutagenesis

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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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.",
keywords = "GTP-binding protein, phenyldiazirine, photoactivatable group, signal transduction, site-specific mutagenesis",
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T2 - Covalent crosslinking of the rhodopsin (metarhodopsin II)-transducin complex - The rhodopsin cytoplasmic face links to the transducin α subunit

AU - Resek, John F.

AU - Farrens, David

AU - Khorana, H. Gobind

PY - 1994/8/2

Y1 - 1994/8/2

N2 - 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.

AB - 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.

KW - GTP-binding protein

KW - phenyldiazirine

KW - photoactivatable group

KW - signal transduction

KW - site-specific mutagenesis

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