Evaluation of the Intrinsic Zn(II) Affinity of a Cys3His1 Site in the Absence of Protein Folding Effects.
Evaluation of the Intrinsic Zn(II) Affinity of a Cys3His1 Site in the Absence of Protein Folding Effects.
复制标题
DOI：
10.1021/acs.inorgchem.5b00718
复制
发表时间：
2015-05
影响因子：
4.6
通讯作者：
Amit R. Reddi;M. Pawlowska;B. Gibney
中科院分区：
文献类型：
--
作者：
Amit R. Reddi;M. Pawlowska;B. Gibney
锌指转录因子是人类基因组中最大的一类金属蛋白。Zn（II）与典型的Cys2His2、Cys3His1或Cys4位点结合，导致金属诱导的蛋白质折叠事件，这是实现其生物活性结构所必需的。然而，金属结合和蛋白质折叠的耦合性质模糊了每个过程对整体锌指稳定的个体自由能贡献。在此，我们使用天然蛋白质支架将金属-配体相互作用的能量贡献从蛋白质-蛋白质相互作用中分离出来，该支架保留了具有和不具有Zn（II）结合的基本相同的结构，Zn（II）结合是人转录因子IIB （ZBD-TFIIB）的59个氨基酸锌结合域。通过荧光法和等温滴定量热法测定了含有单个Cys3His1位点的Zn(II)-ZBD-TFIIB的形成常数为1.5 × 10(15) M（-1）。等温滴定量热法表明，在pH为5.5、7.0和8.0时，锌（II）的结合在熵上有利；在pH为8.0时，锌（II）的结合在焓上有利；在pH为5.5和7.0时，锌（II）的结合在焓上略有不利。比较Zn(II)-ZBD-TFIIB蛋白与天然Cys3His1锌指蛋白的条件离解常数，确定后者中蛋白折叠的自由能成本。我们的分析表明，相对于锌（II）结合的贡献，折叠锌指蛋白的能量成本是最小的，这表明锌（II）结合的真正作用可能是调节蛋白质动力学和/或动态地模板蛋白质折叠过程。
Zinc finger transcription factors are the largest class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys2His2, Cys3His1, or Cys4 sites results in metal-induced protein folding events required to achieve their biologically active structures. However, the coupled nature of metal binding and protein folding obscures the individual free energy contributions of each process toward overall zinc finger stabilization. Herein, we separate the energetic contributions of metal-ligand interactions from those of protein-protein interactions using a natural protein scaffold that retains essentially identical structures with and without Zn(II) bound, the 59 amino acid zinc binding domain of human transcription factor IIB (ZBD-TFIIB). The formation constant of Zn(II)-ZBD-TFIIB, which contains a single Cys3His1 site, was determined to be 1.5 × 10(15) M(-1) via fluorimetry and isothermal titration calorimetry. Isothermal titration calorimetry showed that Zn(II) binding is entropically favored at pH 5.5, 7.0, and 8.0 and enthalpically favored at pH 8.0 but slightly enthalpically disfavored at pH 5.5 and 7.0. The conditional dissociation constants of Zn(II)-ZBD-TFIIB and natural Cys3His1 zinc finger proteins were compared to determine the free energy cost of protein folding in the latter. Our analysis reveals that the energetic cost to fold zinc finger proteins is minimal relative to the contribution of Zn(II) binding and suggests that the true role of Zn(II) binding may be to modulate protein dynamics and/or kinetically template the protein folding process.