Advanced 500 spectrometer

STD NMR Spectrum was recorded using a Bruker Advanced 500 spectrometer equipped with Cryo probes and processed by Bruker® Topspin software. A typical sample preparation consist of 5 μM protein and 100 μM ligand in 5% DMSO-d₆ and deuterated PBS, pH 7.4 buffer (600 ml total volume) for STD NMR experiments. Protein used for STD NMR were tagless MYC (amino acids 353–439) (Cayman Chemicals) and MAX(S) (151 amino acids, purified in this study) (in HEPES – 50 mM, NaCl – 500 mM, B-mercapethonol - 10 mM, Glycerol - 5%, Imidazole - 30 mM). The ligand was tested in the presence and absence of individual proteins (MYC and MAX). The prepared solution was vortexed for 30 secs and submitted for NMR. Various sample preparations tested were Ligand + MYC, Ligand + MAX, G5 + MYC, G5 + MAX, Ligand alone, and G5 alone. STD NMR experiments were performed with a train of 50 ms Gaussian-shaped saturating pulses at 200 Hz power for 2 s with “on” resonance saturation at −2.5 ppm and “off” resonance saturation at 40 ppm. The relaxation delay was 2 s before the saturating pulses. The number of scans was 2048 and the spectral width was 10 ppm.