Topspin v 3.1 or higher

Deuterated solvents were purchased from Deutero GmbH (Kastellaun, Germany). NMR spectroscopic measurements were performed in D₂O at stated temperatures on a Bruker Avance^III 700 MHz (equipped with an inverse 5 mm quadruple-resonance Z-grad cryoprobe). Acetone was used as an external standard for calibration of ¹H (δ_H = 2.225) and ¹³C (δ_C = 30.89) NMR spectra [42 (link)], and 85% of phosphoric acid was used as an external standard for calibration of ³¹P NMR spectra (δ_P = 0.00). All data were acquired and processed by using Bruker TOPSPIN V 3.1 or higher (Bruker BioSpin Corporation, Billerica, MA, USA). ¹H NMR assignments were confirmed by 2D ¹H,¹H-COSY, and total correlation spectroscopy (TOCSY) experiments. ¹³C NMR assignments were indicated by 2D ¹H,¹³C-HSQC, based on the ¹H NMR assignments. Inter-residue connectivity and further evidence for ¹³C assignment were obtained from 2D ¹H,¹³C-heteronuclear multiple bond correlation and ¹H,¹³C-HSQC-TOCSY. Connectivity of phosphate groups were assigned by 2D ¹H,³¹P-HMQC and ¹H,³¹P-HMQC-TOCSY.

The mixtures were freeze-dried and deuterium exchange was accomplished by freeze-drying sample solutions in deuterated water (D₂O; 99.98%, Deutero) twice. NMR spectroscopic measurements were performed at 300 K on a Bruker Avance^III 700 MHz spectrometer equipped with an inverse 5 mm quadruple-resonance Z-grad cryoprobe (spectrometer frequencies: 700.43 MHz for ¹H, 176.12 MHz for ¹³C, and 283.54 MHz for ³¹P). Acetone was used as an external standard for calibration of ¹H (δ_H = 2.225 ppm) and ¹³C (δ_C = 30.89 ppm) NMR spectra; 85% of phosphoric acid was used as an external standard for calibration of ³¹P NMR spectra (δ_P = 0.00 ppm). All data were acquired and processed by using Bruker TOPSPIN V 3.1 or higher (Bruker BioSpin Corporation). The parameter sets used were adapted starting from respective Bruker standard parameter sets, which are all included in this software. The 2D ¹H,³¹P-HMQC-TOCSY spectrum was recorded with a mixing time of 120 ms.