Synthesis and application of bidentate photoaffinity cross-linking reagents: Nucleotide photoaffinity probes with two photoactive groups

Two "targeted bidentate" photoaffinity cross-linking reagents, the monoanhydride of 8-N₃ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([γ-³²P]8-N₃ATPγBP) and the monoanhydride of 8-N₃GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([γ-³²P] 8-N₃GTPγBP), were developed for studying the interand intramolecular interactions of nucleotide-binding proteins. Experiments using these bidentate reagents with two photoactive groups led to specific cross-linking: [γ-³²P]8-N₃GTPγBP and [γ-³²P]8-N₃ATPγBP showed intersubunit cross-linking of glutamate dehydrogenase and [γ-³²P]8-N₃GTPγBP appeared to cross-link the α- and β-subunits of tubulin. The non-azido "monodentate" versions of these reagents, the monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([γ-³²P]ATPγBP) and the monoanhydride of GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([γ-³²P]GTPγBP), were also synthesized and characterized. The ability of these monodentate reagents with one photoactive group to serve as photoaffinity probes was established by photolabeling specifically the exchangeable GTP-binding domain of tubulin with [γ-³²P]GTPγBP and the ATP-binding domain of purified adenylate kinase and several nucleotide-binding proteins in human brain homogenate with [γ-³²P]ATPγBP.