The photoactivatable NAD+ analogue [32P]2-azida-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein Gα1,

R. R. Vaillancourt, N. Dhanasekaran, A. E. Ruoho

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to 'tether' [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the α subunit of the G-protein G(t). Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of α(t) with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the 'acceptor' domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP-ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other α(t) domains and intermolecular interactions with holotransducin α and γ subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2'-nitrophenylsulphenyl)-3-methyl-3'bromoindolenine ('BNPS-skatole') and CNBr. The results indicate that the C-terminus of α(t) interacts with both N-terminal and C-terminal domains within the α(t) molecule. Mapping the interacting sites between cross-linked α dimers and α trimers indicates that the C-terminal domain of α(t) is involved in the formation of α(t) homopolymers in solution. In addition, our studies place the βγ subunit in close proximity to Cys347 of α(t) as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the γ subunit, which was further localized to the C-terminal half of γ(t). The studies presented here identify the C-terminal intra- and inter-molecular interactions of the α subunit of holo-transducin.

Original languageEnglish (US)
Pages (from-to)987-993
Number of pages7
JournalBiochemical Journal
Volume311
Issue number3
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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