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The eukaryotic histone heterodimer H2A–H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol−1, respectively; at 10 μM, the sum of the stability of the monomers is ∼60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding— initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.


Originally published in the Journal of Molecular Biology, 2008; 384(5):1369-83.