32p gtp

ProF (3,6-Diaminoacridine hydrochloride) was purchased from Sigma-Aldrich (MO, USA). TO was purchased from Sigma-Aldrich. C-di-GMP and c-di-AMP were synthesized following literature and obtained as triethylammonium acetate salt [48 (link)]. ³²P-GTP or ³²P-ATP was purchased from Perkin Elmer (MA, USA). Images were obtained by STORM scanner and quantified by ImageQuant software (Molecular Dynamics). Fluorescence was measured by Cary Eclipse Fluorescence Spectrophotometer. AFM image was taken by Veeco Multimode AFM with nanoscope III controller with tapping mode. The visualization probe was Silicon AFM Probes TAP 300 from Ted Pella, Inc. Fluorescence lifetime was measured using time-domain system integrated with fluorescence lifetime imaging microscope (FLIM) system Alba V (ISS, IL, USA). The system is equipped with SPC-830 TCSPC module and pulsed laser system (Becker and Hickl GmbH). Laser BHL-473 nm and observation through band pass filter 514/50 nm was used for TO. Data analysis was performed using Vista Vision software v. 218 from ISS.

[³²P]-labelled c-di-GMP was chemically synthesised using a labelled [³²P]GTP (3000 Ci/mmol, PerkinElmer, USA) and the purified His₆-tagged enzyme tDGC; a protein construct with a key residue mutation (R158A) of diguanylate cyclase from Thermotoga maritime [41 (link)]. Five micromolar tDGC and 20 μCi [³²P]GTP (mixed with 50 μM cold GTP) were added to a mixture of 20 μL reaction buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, and 2 mM DTT). After 3 h at 45°C, the reaction was terminated by heating at 98°C for 10 min. The precipitated protein was removed by centrifugation at 20,000 g for 5 min. Radioactive [³²P]c-di-GMP was tested by separation on a polyethyleneimine-cellulose plate (1:1.5 (v/v) saturated (NH₄)₂SO₄ and 1.5 M KH₂PO₄, pH 3.6). The mixture contained more than 95% [³²P]c-di-GMP without further purification.