Banks Douglas D, Gloss Lisa M
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660, USA.
Biochemistry. 2003 Jun 10;42(22):6827-39. doi: 10.1021/bi026957r.
To compare the stability of structurally related dimers and to aid in understanding the thermodynamics of nucleosome assembly, the equilibrium stabilities of the recombinant wild-type H3-H4 tetramer and H2A-H2B dimer have been determined by guanidinium-induced denaturation, using fluorescence and circular dichroism spectroscopies. The unfolding of the tetramer and dimer are highly reversible. The unfolding of the H2A-H2B dimer is a two-state process, with no detected equilibrium intermediates. The H3-H4 tetramer is unstable at moderate ionic strengths (mu approximately 0.2 M). TMAO (trimethylamine-N-oxide) was used to stabilize the tetramer; the stability of the H2A-H2B dimer was determined under the same solvent conditions. The equilibrium unfolding of H3-H4 was best described by a three-state mechanism, with well-folded H3-H4 dimers as a populated intermediate. When compared to H2A-H2B, the H3-H3 tetramer interface and the H3-H4 histone fold are strikingly less stable. The free energy of unfolding, in the absence of denaturant, for the H3-H4 and H2A-H2B dimers are 12.4 and 21.0 kcal mol(-)(1), respectively, in 1 M TMAO. It is postulated that the difference in stability between the histone dimers, which contain the same fold, is the result of unfavorable tertiary interactions, most likely the partial to complete burial of three salt bridges and burial of a charged hydrogen bond. Given the conservation of these buried interactions in histones from yeast to mammals, it is speculated that the H3-H4 tetramer has evolved to be unstable, and this instability may relate to its role in nucleosome dynamics.
为了比较结构相关二聚体的稳定性，并有助于理解核小体组装的热力学，我们通过胍诱导变性，利用荧光和圆二色光谱法测定了重组野生型H3-H4四聚体和H2A-H2B二聚体的平衡稳定性。四聚体和二聚体的解折叠是高度可逆的。H2A-H2B二聚体的解折叠是一个两态过程，未检测到平衡中间体。H3-H4四聚体在中等离子强度（μ约为0.2 M）下不稳定。使用三甲胺-N-氧化物（TMAO）来稳定四聚体；在相同的溶剂条件下测定H2A-H2B二聚体的稳定性。H3-H4的平衡解折叠最好用三态机制来描述，其中折叠良好的H3-H4二聚体是一个存在的中间体。与H2A-H2B相比，H3-H3四聚体界面和H3-H4组蛋白折叠的稳定性明显较低。在1 M TMAO中，H3-H4和H2A-H2B二聚体在无变性剂时的解折叠自由能分别为12.4和21.0 kcal mol⁻¹。据推测，含有相同折叠的组蛋白二聚体之间稳定性的差异是不利三级相互作用的结果，最有可能是三个盐桥部分至完全埋藏以及一个带电氢键的埋藏。鉴于从酵母到哺乳动物的组蛋白中这些埋藏相互作用的保守性，推测H3-H4四聚体已进化为不稳定，这种不稳定性可能与其在核小体动力学中的作用有关。
