2-azido-[³²P]NAD⁺, a photoactivatable probe for G-protein structure: Evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of α interacts with both α and γ subunits

A radioactive and photoactivatable derivative of NAD⁺, 2-azido-[adenylate-³²P]NAD⁺, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys³⁴⁷ of the α subunit (α_T) of G_T, the retinal guanine nucleotide-binding protein. ADP-ribosylation of α_T followed by light activation of the azide moiety of 2-azido-[adenylate-³²P]ADP-ribose produced four crosslinked species involving the α and γ subunits of the G_T heterotrimer: an α trimer (α-α-α), an α-α-γ crosslink, an α dimer (α-α), and an α-γ crosslink. The α trimer, α-α-γ complex, α dimer, and α-γ complexes were immunoreactive with α_T antibodies. The α-α-γ and the α-γ complexes were immunoreactive with antisera recognizing γ subunits. No evidence was found for crosslinking of α_T to β_T subunits. Hydrolysis of the thioglycosidic bond between Cys³⁴⁷ and 2-azido-[adenylate-³²P]ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys³⁴⁷ of α_T in the crosslinked oligomers to α monomers, indicative of intermolecular photocrosslinking, and to γ monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that G_T exists as an oligomer and that ADP-ribosylated Cys³⁴⁷, which is four residues from the α_T carboxyl terminus, is oriented toward and in close proximity to the γ subunit.