Gloss Lisa M, Placek Brandon J
School of Molecular Biosciences, Washington State University, Pullman 99164-4660, USA.
Biochemistry. 2002 Dec 17;41(50):14951-9. doi: 10.1021/bi026282s.
The core nucleosome, which comprises an H3-H4 tetramer and two H2A-H2B dimers, is not a static DNA packaging structure. The nucleosome is a dynamic protein-DNA complex, and the modulation of its structure is an important component of transcriptional regulation. To begin to understand the molecular details of nucleosome dynamics, we have investigated the stability of the isolated H2A-H2B dimer. The urea-induced equilibrium responses of the heterodimer have been examined by far-UV circular dichroism and intrinsic tyrosine fluorescence. The two spectroscopic probes yielded coincident transitions, and global fitting of the reversible urea-induced unfolding further demonstrated that H2A-H2B unfolds by a two-state equilibrium response. At physiological ionic strengths, the free energy of unfolding in the absence of urea of H2A-H2B is 11.8 +/- 0.3 kcal mol(-)(1), moderate stability for a dimer of 26.4 kDa. The m value, or sensitivity of the unfolding to urea, is 2.9 +/- 0.1 kcal mol(-)(1) M(-)(1). This value is significantly larger than would be predicted for the unfolding of the dimerization motif alone ( approximately 2 kcal mol(-)(1) M(-)(1)), suggesting that the N-terminal tails may adopt a collapsed, solvent-excluding structure that undergoes an unfolding transition. The efficacies of several potassium salts and three chloride salts to stabilize the H2A-H2B dimer were determined. The salt-dependent stabilization of the H2A-H2B dimer shows that the Hofmeister effect is the predominant mode of stabilization. However, studies employing multiple salts suggest that there is a component of stabilization that must arise from screening of electrostatic repulsion in the highly basic heterodimer. The most highly charged regions of the dimer are the N-terminal tails, sites of posttranslational modifications such as acetylation and phosphorylation. These modifications, which alter the charge density of the tails, are involved in regulation of nucleosome dynamics.
核心核小体由一个H3-H4四聚体和两个H2A-H2B二聚体组成，它不是一个静态的DNA包装结构。核小体是一种动态的蛋白质-DNA复合物，其结构的调节是转录调控的重要组成部分。为了开始了解核小体动力学的分子细节，我们研究了分离出的H2A-H2B二聚体的稳定性。通过远紫外圆二色性和内在酪氨酸荧光检测了异二聚体的尿素诱导平衡响应。这两种光谱探针产生了一致的转变，对可逆尿素诱导的去折叠进行的全局拟合进一步表明，H2A-H2B通过双态平衡响应去折叠。在生理离子强度下，H2A-H2B在无尿素时的去折叠自由能为11.8±0.3千卡摩尔⁻¹，对于一个26.4 kDa的二聚体来说稳定性适中。m值，即去折叠对尿素的敏感性，为2.9±0.1千卡摩尔⁻¹ M⁻¹。这个值明显大于仅二聚化基序去折叠所预测的值（约2千卡摩尔⁻¹ M⁻¹），这表明N端尾巴可能采取一种折叠的、排除溶剂的结构，该结构会经历去折叠转变。测定了几种钾盐和三种氯盐稳定H2A-H2B二聚体的效果。H2A-H2B二聚体的盐依赖性稳定表明霍夫迈斯特效应是主要的稳定模式。然而，使用多种盐的研究表明存在一种稳定成分，它必定源于对高度碱性异二聚体中静电排斥的屏蔽。二聚体中电荷最高的区域是N端尾巴，即翻译后修饰如乙酰化和磷酸化的位点。这些修饰改变了尾巴的电荷密度，参与了核小体动力学的调控。
