Identification of peptide binding sequence of TRIM25 on 14-3-3σ by bioinformatics and biophysical techniques

In Silico Prediction and Biophysical Validation of Novel 14-3-3σ Homodimer Stabilizers

“…Peptides 3 , 5 , 9 , and 16 were prepared in 100 mM phosphate buffer, 50 mM NaCl, 10% (v/v) 2 H 2 O, pH 7. CPMG experiments were recorded on a Bruker NMR 500 MHz spectrometer, equipped with a BBO probe, for each peptide (1 mM) in the presence and absence of 14-3-3σ protein (50 μM), as described previously, with a spin-lock time of 300 ms, number of scans = 32 scans, dummy scans = 8 scans, spectral width = 8012.82 Hz, delay between 180° pulses in the CPMG = 1 ms, and relaxation delay (D1) = 4 s. The competition [ 1 H]-CPMG NMR assay was conducted using the same parameters with 1 mM peptide 3 , 1 mM ExoS peptide, and 50 μM 14-3-3σ protein. TOPSPIN software, version 3.2; Bruker Corporation: Billerica, Massachusetts, US, 2015, and TOPSPIN software, version 4.1; Bruker Corporation: Billerica, Massachusetts, US, 2020 were used for data acquisition and analysis, respectively.…”