(A) Overlay of a region of the 1H-15N HSQC (or TROSY-HSQC) spectra of 50 μM 15N-BPTF PHD upon titration of H3(1–10)K4me3 (left), H3KC4me3-NCP (center), or H3(1–44)KC4me3 (right). Spectra are color-coded according to ligand concentration, with apo spectra in black and shades of salmon for increasing concentrations of ligand. Spectra were collected at molar ratios (PHD:total H3KC4me3 mark) of: 1:0, 1:0.1, 1:0.25, 1:0.5, 1:1, 1:2, and 1:5 for H3(1–10)K4me3, 1:0, 1:0.2, 1:0.5, 1:1, 1:2, 1:4 and 1:6 for H3KC4me3-NCP, and 1:0, 1:0.1, 1:0.25, 1:0.5, 1:1, 1:2, 1:5 and 1:10 for H3(1–44)KC4me3. Dashed arrows track the trajectory between the apo and bound states for the two peptide titrations (in black). The H3(1–44)KC4me3 trajectories are shown (in grey) to compare titration trajectories. To account for the TROSY effect, the displayed region for the NCP titration is shifted by JNH/2 Hz. Data was collected at 37°C in 150 mM KCl. (B) Surface representation of the structure of H3K4me3-bound BPTF PHD finger (PDB ID 2F6J) with residues that are significantly perturbed upon binding to H3(1–44)KC4me3 or H3KC4me3-NCP colored in shades of salmon (average +2, +1, +1/2 standard deviations colored in raspberry, deep salmon, and wheat, respectively). The H3 tail peptide is colored pale gold and shown in stick representation. (C) CSPs (Δδ) corresponding to (A) are plotted as a function of residue for H3(1–10)K4me3 (open bars), H3KC4me3-NCP (filled black bars), or H3(1–44)KC4me3 (grey bars) for the highest ligand concentration reached in each of the titrations. As expected, the largest CSPs were observed for residues within and neighboring the aromatic cage (Y2876-E2878, F2881, Y2882, and W2891) where the K4me3 group binds, and the binding pockets for H3A1 (D2908) and H3R2 (R2887-Q2889). Note that the titration with H3KC4me3-NCP did not reach saturation. The * indicates a residue that broadens beyond detection in the fully bound state. The *** indicates a residue that is significantly perturbed along the course of the titration but broadens beyond detection in the fully bound state. An ‘X’ indicates that a residue is either not observable or not assigned. The Δδ for the H3KC4me3-NCP titration is shown re-scaled below for ease of visualization.