The insoluble histones were re-dissolved in 4 ml of unfolding Buffer (7 M Guanidinium-HCl, 20 mM HEPES-KOH pH 7.5, 1 mM EDTA, 1 mM click here DTT) and dialysed into SAU200 Buffer (20 mM sodium acetate pH 5.2, 7 M urea, 200 mM NaCl, 1 mM EDTA, 5 mM β-mercaptoethanol). 0.5 ml of cation exchange resin (SP FF, GE Healthcare) was equilibrated with SAU200 buffer in 10 mL disposable chromatography columns (Bio-Rad). Dialysed histones were bound to the resin, washed twice with 2 mL of SAU200, once with 2 mL of SAU400 (400 mM NaCl), and

eluted in 2 mL of SAU800 (800 mM NaCl). Eluted histones were dialysed into H2O plus 5 mM β-mercaptoethanol and lyophilized. Histones were re-dissolved in unfolding Buffer, quantified by absorbance at 280 nm and mixed in equimolar amounts. The octamer complex was refolded by dialysis into refolding buffer (2 M NaCl, 20 mM HEPES-KOH pH 7.5, 1 mM EDTA, 5 mM β-mercaptoethanol), and purified from mis-folded aggregates by gel filtration on a GL 10/300 column packed with Superdex S200(GE Healthcare). Before gel filtration, 20 mM dithiothrietol was added to the samples and incubated at 25 °C for 30 min to

ensure complete reduction of the histone H3 labeling site. Gel filtration was carried out in Refolding Buffer without β-mercaptoethanol. Immediately after gel filtration, fractions containing the correctly folded histone octamer were concentrated, using an Amicon Ultra-4 selleck products centrifugal concentrator (Millipore) with a molecular weight cut off of 10,000 Da, to ∼25 μM, and spin labeled with a ten-fold excess of non-deuterated (1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL) (Fig. S2) at 25 °C for 3 h. Excess MTSSL was removed by dialysis verses 2 L of refolding buffer without reducing agents at 4 °C for 16 h. Labeled octamer was combined with a 1-fold excess of H2A–H2B dimers,

refolded and purified separately, as our previous work had shown that an excess of dimer stabilizes the octamer complex [10]. H2O in the samples was exchanged for D2O by four rounds of sequential concentration Nintedanib (BIBF 1120) and dilution, with deuterated refolding buffer minus reducing agent (prepared by lyophilisation and re-solvation of buffer with D2O), using Amicon Ultra-4 centrifugal concentrators (Millipore), achieving 99.8% exchange with D2O. The octamer samples were finally concentrated to 50 μM and diluted 1:1 with D8-glycerol (Cambridge Isotope Laboratories Inc.), giving a final spin-pair concentration of approximately 25 μM, and stored at 4 °C until EPR measurements were made. Solvent exchange and subsequent sample preparation steps took approximately 2.5 h at room temperature and subsequently samples were routinely stored at 4 °C for several days. Based on reported hydrogen–deuterium exchange rates in proteins [13] and the inherent structural lability of the core histone octamer, it was expected that almost complete exchange of protons would have been achieved.