Fig. 6: Biochemical analysis of peptide conformation upon binding to SETD2 and peptide methylation experiments.

a Schematic representation of EDANS-H3K36-Dabcyl and EDANS-ssK36-Dabcyl hairpin conformation unfolding upon binding to SETD2 leading to an increase in the N-C distance and reduced FRET. b EDANS-H3K36-Dabcyl (1 µM) and EDANS-ssK36-Dabcyl (1 µM) were dissolved in buffer and the fluorescence was monitored. After 20 min, SETD2 (2 µM) was added to each peptide resulting in an intensity increase of the fluorescence. To monitor fluorescence changes due to the changes in buffer volume and concentrations, control experiments were conducted with addition of buffer to the peptides instead of SETD2. The fluorescence signal of peptides with addition of SETD2 was expressed relative to the signal with addition of buffer. c Peptides without quencher were used as a negative control to ensure that FRET was the reason for the fluorescence intensity increase observed in panel b. In each panel, the results of two independent experiments are shown. d, e SPOT peptide array methylation assay to investigate the effect of an enforced hairpin conformation of peptide substrates on SETD2 activity. H3K36 and ssK36 were prepared on a SPOT array together with derivates in which P30 and P43 were altered to C, designated by (C-C). The two Cys residues allow the formation of a disulphide bond that arrests the peptides in the hairpin conformation. The peptide array was then methylated by SETD2 using radioactively labelled SAM and detected by autoradiography. Panel d shows one example of an autoradiography image after 2 weeks of film exposure. The two lanes represent technical duplicates and contain identical peptides. Panel e shows the average of three independent experiments each containing a technical duplicate set of all spots and the error bars represent the SEM. Signals were normalised to the average signal of all spots in each experiment. P-values were determined by t-test for a two-tailed distribution of paired values.