Mutational studies uncover non-native structure in the dimeric kinetic intermediate of the H2A-H2B heterodimer.
Mutational studies uncover non-native structure in the dimeric kinetic intermediate of the H2A-H2B heterodimer.
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突变研究揭示了 H2A-H2B 异二聚体的二聚动力学中间体中的非天然结构。
DOI：
10.1016/j.jmb.2010.06.034
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发表时间：
2010
影响因子：
5.6
通讯作者：
Gloss,LisaM
中科院分区：
文献类型：
--
作者：
Stump,MatthewR;Gloss,LisaM
组蛋白H_2A-H_2B异源二聚体的折叠途径最小限度地包括一个在途径上的二聚体，突变期中间体I2。处于平衡状态的部分折叠的H_2A和H_2B单体被表征为潜在的单体动力学中间体。比较了从分离的折叠单体和在4M尿素中展开的杂二聚体引发折叠的动力学。在0.4M尿素以上，观察到的速率几乎相同，表现出与最终变性剂浓度的对数线性关系。低于∼0.4M尿素(从4-M尿素展开状态无法获得的浓度)，观察到速率的滚动；这表明I2系综的一个组分包含重新排列/异构化为更像本地的物种的非天然结构。通过一组包含9个位点的Ala和Gly替换的H_2A-H_2B突变体，主要集中在长的中央α2螺旋上，评估了螺旋倾向对I2系综稳定性的贡献。收集平衡和动力学折叠/去折叠数据，以确定突变对I2稳定性和I2和N2之间过渡态的影响。这一有限的突变研究表明，H_2A和H_2B的α_2螺旋以及H_2B的α_1和H_2A的α_3的C末端和αC短螺旋中的残基对I2猝发相物种的稳定性有贡献。有趣的是，九个靶向残基中至少有八个通过某种程度上非天然的相互作用稳定了I2。鉴于不稳定的I2和这些非天然相互作用不会加速折叠，因此得出结论，I2系综中存在的天然和非天然结构能够有效地折叠H2A-H2B。
The folding pathway of the histone H2A–H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I2. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below ∼0.4 M urea (concentrations inaccessible from the 4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I2ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I2ensemble was assessed with a set of H2A–H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central α2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I2and the transition state between I2and N2. This limited mutational study indicated that residues in the α2 helices of H2A and H2B as well as α1 of H2B and both the C-terminus of α3 and the short αC helix of H2A contribute to the stability of the I2burst-phase species. Interestingly, at least eight of the nine targeted residues stabilize I2by interactions that are non-native to some extent. Given that destabilizing I2and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structures present in the I2ensemble enable efficient folding of H2A–H2B.