Structure and function in rhodopsin. Single cysteine substitution mutants in the cytoplasmic interhelical E-F loop region show position- specific effects in transducin activation

Ke Yang, David Farrens, Wayne L. Hubbell, H. Gobind Khorana

Research output: Contribution to journalArticle

96 Citations (Scopus)

Abstract

The cytoplasmic interhelical E-F loop in rhodopsin is a part of the region that interacts with the G protein transducin and rhodopsin kinase during signal transduction. In extending the previous work on systematic single cysteine substitutions of the amino acids in the cytoplasmic C-D loop, we have now replaced, one at a time, the amino acids Q225-1256 in the E-F loop region by cysteines. All the mutants formed the characteristic rhodopsin chromophore with 11-cis-retinal. While most of the mutants bleached normally, L226C, showed abnormal bleaching behavior. A study of the alkylation of the mutants by N-ethylmaleimide in dark showed low reactivity by some mutants, especially L226C. The rates of transducin activation (Gm(T(α))-GTPγS complex formation) were measured for all the mutants. While these were normal for the bulk of the mutants, some (1.226C, T229C, V230C, A233C, A234C, T242C, T243C, and Q244C) showed strikingly reduced transducin activation. The results suggest a specific structure in the E F loop that interacts with transducin.

Original languageEnglish (US)
Pages (from-to)12464-12469
Number of pages6
JournalBiochemistry
Volume35
Issue number38
DOIs
StatePublished - 1996
Externally publishedYes

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Transducin
Rhodopsin
Cysteine
Substitution reactions
Chemical activation
G-Protein-Coupled Receptor Kinase 1
Retinaldehyde
Amino Acids
Signal transduction
Ethylmaleimide
Alkylation
Amino Acid Substitution
Chromophores
Bleaching
GTP-Binding Proteins
Protein Kinases
Signal Transduction

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure and function in rhodopsin. Single cysteine substitution mutants in the cytoplasmic interhelical E-F loop region show position- specific effects in transducin activation. / Yang, Ke; Farrens, David; Hubbell, Wayne L.; Gobind Khorana, H.

In: Biochemistry, Vol. 35, No. 38, 1996, p. 12464-12469.

Research output: Contribution to journalArticle

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