The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein G alpha t.
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 alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha 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 alpha t domains and intermolecular interactions with holotransducin alpha and gamma 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 alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000737:chemistry*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009243:NAD / Q000031:analogs & derivatives* / Q000737:chemistry; D010449:Peptide Mapping; D010761:Phosphorus Radioisotopes; D010777:Photochemistry; D010782:Photolysis; D015293:Transducin / Q000737:chemistry
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D009243:NAD; C062879:nicotinamide 2-azidoadenine dinucleotide; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; D019204:GTP-Binding Proteins; D015293:Transducin