Quantification of cAMP in CNBD Proteins

Determination of the cAMP content of purified CNBD proteins performed following the procedure previously described^{13 (link)}. Endogenous cAMP bound to His₆-MBP tagged HCN1/2 CNBDs purified from Escherichiacoli Rosetta strain (EMD Millipore) was released by boiling the protein sample (∼1 mg) for 2 min. The boiled protein samples were centrifuged at maximum speed for 10 min, at room temperature, and the supernatant was diluted in 5 mM ammonium bicarbonate. The sample was loaded onto an anion exchange chromatography column (HiTrapQ (1 ml), GE Healthcare), previously equilibrated with 5 mM ammonium bicarbonate. After a washing step (7 column volumes) with 5 mM ammonium bicarbonate, cAMP was eluted with a linear gradient of ammonium bicarbonate (5–1000 mM) in 20 column volumes. Calibration curves were performed by loading in the HiTrapQ column 5, 10 and 20 nmoles of cAMP diluted in 5 mM ammonium bicarbonate. Chromatographies were performed at 4 °C and monitored using the AKTApurifier UPC 10 fast protein liquid system (GE Healthcare). Calculation of the area of the peaks of cAMP eluted from the HiTrapQ column was performed by using the software UNICORN5.11.