NMR analysis of EGCG–Uba1 binding

NMR spectra were acquired with a Bruker Avance 600 MHz spectrometer equipped with a 5 mm z-gradient CP-TCI triple-resonance cryoprobe at 303 K. EGCG and Uba1 were dissolved in 20 mM phosphate-buffered saline at pH 7.4 (90% H₂O, 10% D₂O), containing 150 mM NaCl and 0.02% NaN₃. In order to prevent EGCG oxidation, 1 mM tris(2-carboxyethyl)phosphine was added to the solutions. EGCG and Uba1 concentrations were 400 μM and 4 μM, respectively. All the spectra were acquired with excitation sculpting solvent suppression pulse scheme. In STD experiments, 40 equally spaced 50 ms Gaussian-shaped pulses were used for the selective saturation of the protein, thus the total saturation time was 2 s. The on-resonance irradiation and the off-resonance saturation frequency were set at 0.86 ppm and 40.0 ppm, respectively. A total of 2k scans were collected for each pseudo 2D experiment. 2D NOESY experiments were acquired with 128 increments and a mixing time of 100 ms. As a control, all the experiments were repeated for a sample containing EGCG at the same concentration but in the absence of the protein.