Histone peptide binding assay for PfBDP1-BRD

Chemical shift perturbation experiments were performed using 0.3–0.5 mM of uniformly ¹⁵N-labeled PfBDP1-BRD (303–488) in NMR buffer containing 20 mM Tris-HCl pH 6.8, 150 mM NaCl, 10 mM DTT and 10% D₂O. Titration mixtures of the PfBDP1-BRD (303–488) and each of the modified histone peptides (res 1–24) unmodified H3, H3K9ac, H3K14ac, H3K9acK14ac, unmodified H4, H4K5ac, H4K12ac, H4K5acK8acK12acK16ac, unmodified H2A.Z, H2A.ZK11ac, unmodified H2B.Z, H2B.ZK3acK8acK13acK14acK18ac were prepared at molar ratios of 1:0, 1:0.5, 1:1, 1:2.5, and 1:5 M, in a total volume of 80 μL, The sample mixtures were then transferred into 1.7 mm NMR tubes (Bruker). Two-dimensional ¹⁵N−¹H HSQC (heteronuclear single quantum coherence) experiments for all samples were acquired on Bruker Avance III spectrometer equipped with a cryogenic 1.7 mm probe. The temperature of the samples was regulated at 25°C throughout data collection. All spectra were processed using TopSpin (Bruker) as described previously [43 (link)] and analyzed using NMRFAM-SPARKY [48 (link)].