Hepes d18

Ligand binding was detected by STD NMR spectroscopy. The binding buffer contained 25 mM Hepes-d18 (Cambridge Isotope Laboratories), pH 7.3, 150 mM NaCl, 10 mM MgCl₂, 2 mM TCEP, 5 μM full-length PKG1α (human PKG1α_2-671) or isolated regulatory domain (human PKG1α_79-356) with and without ligands (cGMP, cAMP, or piperidine series activators), and 25 μM TSP in 99.98% D₂O. All STD NMR spectra were collected at 298 K on a Bruker 600 MHz Avance spectrometer. Selective saturation of the protein was applied by switching the on- and off-resonance saturation frequency after each scan. A train of Gaussian shape pulses with 50 ms pulse length (corresponding to an excitation width of 100 Hz) separated by a delay of 1 ms was used, with the total length of the selective saturation set to 3 s, and the on- and off-resonance saturation frequencies set to −120 and 20,000 Hz. The total time to acquire one STD NMR dataset, including time to change samples, was 50 min.

The NMR samples were prepared in 20 mM HEPES‐d₁₈ (pH 6.8), which was obtained from the Cambridge Isotope Laboratories (Tewksbury, MA). The samples contained 10% D₂O and 0.2 mM DSS, both purchased from the Cambridge Isotope Laboratories. The concentration of the Lmod2 EDRR was 0.3 mM. The sample was titrated with small aliquots of 10 mM and 30 mM CaCl₂ or MgCl₂ stock solutions (adjusted to pH ~6.8). 1H NMR spectra were recorded at 25°C on a Varian Inova 500 spectrometer (500 MHz) equipped with a 5 mm triple‐resonance probe. The spectra were processed, visualized, and analyzed with Mnova (Mestrelab Research, Santiago de Compostela, Spain).