For PKA, the expression and purification was performed as follows: the PKA construct was ordered from Addgene, PCR-amplified with an N-terminal His 9 -tag and an HRV 3C-cleavage site and cloned into the pET15b plasmid with NcoI/BamHI restriction site. The construct was then transformed into chemically competent Escherichia coli Rosetta2 (DE3) and expressed. Purification of the target protein was then performed by Ni affinity chromatography, dialysis with PreScission protease, reverse Ni affinity chromatography, cation exchange and finally size exclusion chromatography. Afterwards, PKA was concentrated to 15 mg/mL, frozen in liquid nitrogen and stored at -80°C until it was used again. Prior to ITC measurements, the required amount of PKA was thawed and purified again via a size exclusion chromatography column and concentrated to approximately 10 mg/mL in 30 mM HEPES, 100 mM NaCl, 10 mM MgCl 2 at pH 7.4.