Binding epitopes mapping by STD NMR

STD spectra were recorded at 25 °C on a Bruker 600 MHz NMR spectrometer. NMR data were processed and analyzed using Topspin 3.5pl7, Topspin 4.0.6, and Mnova 12.0.3. 1 mM EGCG was mixed separately with 5 μM full-length p53 and 10 μM p53 NTD, dissolved in 20 mM Tris–HCl, 150 mM NaCl, 1 mM TCEP at pH 7.2 in 90/10% H₂O/D₂O. 1D STD-NMR spectra were recorded with 256 scans and selective saturation of protein resonances at 1 ppm using a series of Gaussian-shaped pulses, for a total saturation time of 2.0 s. A 30 ms spin lock was also employed to suppress p53 signals that overlap EGCG resonances. Saturation transfer reference (STR) spectrum was recorded with off-resonance frequency set to −10 ppm. STD spectrum of 1 mM EGCG alone without any protein was conducted under the same parameters as a negative control. STD amplification factors (STD_af) of each EGCG proton peak was calculated as STDaf=ISTDI0×[EGCG]T[P], where I_STD represents the signal intensity in the STD spectrum, I₀ is the peak intensity in the STR spectrum, [EGCG]_T is the total EGCG concentration and [P] is the total protein concentration. Normalized STD_af was calculated for each involved proton with the highest intensity signal set to 100%³³.