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

[Image: see text] Zinc finger proteins that bind Zn(II) using a Cys(2)His(2) 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 Cys(2)His(2)/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, 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 Cys(2)His(2) residues, and corresponding potentiometric titrations of Zn(II)–WT1-4 followed by fluorescence spectroscopy yielded the effective pK(a)(eff) values of the Cys(2)His(2) 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)–WT1-4, K(f)(ML) value of 7.5 × 10(12) M(–1), with a limiting K(d) value of 133 fM. This shows that Zn(II) binding to the Cys(2)His(2) 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–Cys(2)His(2) 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 ≤+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, −17.6 kcal/mol.