Acetylation of Histone H3 K56

The histone proteins were expressed and purified as previously described.^{21 (link)} H3K56C/C110A double mutant was used for acetylating histone H3 in a fully unfolded state. Acetylation at H3K56 (H3K56ac) was through thiol–ene coupling between cysteine thiol and N-vinylacetamide (NVA), which is functionally equivalent to the natural acetylation at histone.²² See Figure S1 for the mass-spectrometric analysis to confirm acetylation. The H3 K56C/C110A double mutant was dissolved in a buffer of 200 μL total volume containing 0.2 M sodium acetate (pH 4), 6 M guanidine–HCl, 7 mM L-glutahione, 50 mM N-vinylacetamide, 100 mM dimethyl sulfide, and 5 mM VA-044 (2,2′-[azobis(dimethylmethlene)]bis(2-imidazoline)-dihydrochoride), resulting in the final protein concentration of 1 mM. The reaction mixture was incubated for 2 h at 70 °C and dialyzed against deionized water and then lyophilized overnight for storage at −80 °C.