Characterization of the Zn(II) Binding Properties of the Human Wilms鈥?Tumor Suppressor Protein C-terminal Zinc Finger Peptide

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• 作者：Ka Lam Chan ; Inna Bakman ; Amy R. Marts ; Yuksel Batir ; Terry L. Dowd ; David L. Tierney ; Brian R. Gibney
• 刊名：Inorganic Chemistry
• 出版年：2014
• 出版时间：June 16, 2014
• 年：2014
• 卷：53
• 期：12
• 页码：6309-6320
• 全文大小：464K
• 年卷期：v.53,no.12(June 16, 2014)
• ISSN：1520-510X

文摘

Zinc finger proteins that bind Zn(II) using a Cys₂His₂ coordination motif within a 尾尾伪 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 尾尾伪 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/尾尾伪 zinc finger domain, the C-terminal finger of the Wilms鈥?tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, K_d 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 pK_a values of the Cys₂His₂ residues, and corresponding potentiometric titrations of Zn(II)鈥?b>WT1-4 followed by fluorescence spectroscopy yielded the effective pK_a^eff values of the Cys₂His₂ ligands bound to Zn(II). The K_d, pK_a, and pK_a^eff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)鈥?b>WT1-4, K_f^ML value of 7.5 脳 10¹² M^鈥?, with a limiting K_d value of 133 fM. This shows that Zn(II) binding to the Cys₂His₂ site in WT1-4 provides at least 鈭?7.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)鈥?b>WT1-4 to those from the model peptide Zn(II)鈥?b>GGG鈥揅ys₂His₂ over the pH range 5.0 to 9.0 and a comparison of their pH-independent K_f^ML 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 鈮?4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, 鈭?7.6 kcal/mol.