A total of six independent assays were considered for the analyses. In four assays, proteins were allowed to interact with GlnZ in the presence of ATP and were eluted in the presence of ATP plus 2-OG (namely, ATP1 to ATP4). In two assays, proteins were allowed to interact with GlnZ in the presence of ADP and were eluted in the presence of ATP plus 2-OG (namely, ADP1 and ADP2).
Assays ATP1 and ATP2 were performed using Ni2+ HiTrap chelating columns (GE Healthcare), assay ATP3 was performed using Protino Ni-IDA column (Macherey-Nagel), and assay ATP4 was performed using anti-FLAG M2 magnetic beads (Sigma-Aldrich). The rationale of using different chromatographic matrices was to discard proteins that could be enriched due to spurious affinity to the matrix. Both ADP1 and ADP2 assays were performed using Ni2+ HiTrap chelating columns (GE Healthcare).
Proteins eluted from His-tagged GlnZ Ni2+ affinity columns or FLAG-tagged GlnZ affinity columns were analyzed by label-free LC-MS/MS as described previously (61 (link)) (details in Text S1).
Gerhardt E.C., Parize E., Gravina F., Pontes F.L., Santos A.R., Araújo G.A., Goedert A.C., Urbanski A.H., Steffens M.B., Chubatsu L.S., Pedrosa F.O., Souza E.M., Forchhammer K., Ganusova E., Alexandre G., de Souza G.A, & Huergo L.F. (2020). The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense. mSystems, 5(6), e00817-20.
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