Synthesis and utilization of 8-azidoguanosine 3'-phosphate 5'-[5'-32P]phosphate. Photoaffinity studies on cytosolic proteins of Escherichia coli.

A family of guanosine 3',5'-phosphorylated nucleotides have been postulated to have pleiotypic regulatory properties in prokaryotes during the stringent response. To study proteins which may interact with nucleotides of this homologous series, a photoactive analog of guanosine 3',5'-diphosphate has been synthesized. The analog, 8-azidoguanosine 3'-phosphate 5'-[5'-32P]phosphate, proved to be an effective photoaffinity probe for two nucleotide-binding proteins of Escherichia coli sonicates. It predominately photolabels two proteins with approximate molecular weights of 86,000 and 65,000 (p86 and p65, respectively). The Kd for p65 was approximately 10 microM; that for p86 was not determined. The nucleotide-binding sites were characterized by photolabeling in the presence of various nucleotides. The nucleotides guanosine 3',5'-dipyrophosphate, guanosine 3'-monophosphate 5'-diphosphate, and GTP were most effective at decreasing photoincorporation into p86; guanosine 3'-diphosphate 5'-monophosphate was least effective, with guanosine 3',5'-diphosphate and GMP having an intermediate effect. ATP increased photolabeling of p86. However, ATP was one of the best of the nucleotides studied at decreasing photolabeling of p65, although guanosine 3'-monophosphate 5'-diphosphate, guanosine 3',5'-diphosphate, and GMP appeared only slightly less effective. The relative lack of effectiveness of guanosine 3'-diphosphate 5'-monophosphate inhibiting photolabeling of either protein supports observations that this nucleotide does not have a regulatory role in E. coli. The results presented indicate that the 8-azidoguanosine analogs of this homologous series will prove to be effective probes for studying the protein-nucleotide interactions involved in the stringent response.