Specific and high-affinity interaction of PSIP1-PWWP with H3K
36me3 nucleosomes. (a) Sections of 2D 13C-1H methyl-TROSY spectra focusing on the resonances of the H3V35 methyl groups. Black spectrum corresponds to free nucleosomes and red to 1:2.7 molar ratio. PWWP ILV methyl groups are indicated with green labels. H3V35a/b refer to either of the V35 γ1/γ2 methyl groups. (b) Experimental (points) and fitted (solid lines) line shapes of H3V35a resonance, taken through the peak maximum in the 13C dimension, at the indicated molar ratios of nucleosome:PWWP. Best fit KD is 1.5 μM (95% probability KD < 8 μM) and koff is 500 s-1 (95% probability 400 <koff < 1000 s-1). (c) Line shape-derived binding curve, highlighting the saturation of the binding sites. Change in H3V35a peak position is plotted as a function of PWWP domain added; points - experimental values, lines - fitted values. (d) EMSA-based binding curves and fits for two independent titrations of H3KC36me3 nucleosomes with GST-PWWP (left), along with the ethidium-stained gels (right). Blue/green/red - integrated density of free/+1 PWWP/+2 PWWP nucleosome band. Estimated KD values are indicated.