Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide.
Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide.
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DOI：
10.1021/ic500862b
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发表时间：
2014-06-16
影响因子：
4.6
通讯作者：
Gibney, Brian R.
中科院分区：
文献类型：
--
作者：
Chan, Ka Lam;Bakman, Inna;Marts, Amy R.;Batir, Yuksel;Dowd, Terry L.;Tierney, David L.;Gibney, Brian R.
锌指蛋白（Zinc finger protein）是真核生物系统中最丰富的DNA结合转录因子结构域，它通过ββα蛋白折叠内的Cys 2 His 2配位基序与Zn（II）结合。这些经典的锌指通常在apo状态下未折叠，并且在掺入Zn（II）后自发折叠成其功能性ββα折叠。这些金属诱导的蛋白质折叠事件通过耦合蛋白质折叠和金属离子结合热力学来掩盖蛋白质折叠的自由能成本。在此，我们确定了Cys 2 His 2/ββα锌指结构域的形成常数，该锌指结构域是Wilms肿瘤抑制蛋白（WT 1 -4）的C-末端指，以确定其蛋白质折叠的自由能成本。使用荧光光谱法通过直接滴定或竞争滴定测定pH值为5至9时的单个条件解离常数Kd值。apo-WT 1 -4的电位滴定，然后NMR光谱提供了Cys 2 His 2残基的固有pKa值，并且Zn（II）-WT 1 -4的相应电位滴定，然后荧光光谱产生了与Zn（II）结合的Cys 2 His 2配体的有效pKaeff值。将Kd、pKa和pKaeff值合并到最小完全平衡模型中，得到Zn（II）-WT 1 -4的pH非依赖性形成常数值，KfML值为7.5 × 1012 M-1，极限Kd值为133 fM。这表明Zn（II）与WT 1 -4中的Cys 2 His 2位点结合提供了至少-17.6 kcal/mol的驱动力来折叠蛋白质支架。Zn（II）-WT 1 -4与模型肽Zn（II）-GGG-Cys 2 His 2在pH 5.0至9.0范围内的条件解离常数的比较以及它们的pH非依赖性KfML值的比较表明，WT 1 -4中蛋白质折叠的自由能成本小于+2.1 kcal/mol。这些结果验证了我们的GGG模型系统用于确定天然锌指蛋白中蛋白质折叠的成本，并支持大多数锌指蛋白中蛋白质折叠的成本≤+4.2 kcal/mol的结论，这一值与Zn（II）结合的自由能贡献（−17.6 kcal/mol）相比相形见绌。利用一系列平衡测量方法测定了天然Cys 2 His 2 ββα锌指肽的形成常数。将这些数据与Zn（II）− Cys 2 His 2蛋白事件的文献值进行比较，以评估具有金属诱导的蛋白质折叠事件的蛋白质中蛋白质折叠的自由能成本。的数据导致的结论是，锌（II）结合的自由能的贡献是远远超过蛋白质折叠的最小自由能成本。
Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ββα protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ββα folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/ββα zinc finger domain, the C-terminal finger of the Wilms’ tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)–WT1-4 followed by fluorescence spectroscopy yielded the effective pKaeff values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKaeff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)–WT1-4, KfML value of 7.5 × 1012 M–1, with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least −17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)–WT1-4 to those from the model peptide Zn(II)–GGG–Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent KfML values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is ≤+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, −17.6 kcal/mol. The formation constant for a natural Cys2His2 ββα zinc finger peptide has been determined using a suite of equilibrium measurements. These data are compared to literature values for Zn(II)−Cys2His2 protein events to assess the free energy cost of protein folding in proteins with metal-induced protein folding events. The data lead to the conclusion that the free energy contribution of Zn(II) binding is far in excess of the minimal free energy cost of protein folding.
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