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Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide.

Chan KL, Bakman I, Marts AR, Batir Y, Dowd TL, Tierney DL, Gibney BR - Inorg Chem (2014)

Bottom Line: 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 Kf(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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Brooklyn College , 2900 Bedford Avenue, Brooklyn, New York 11210, United States.

ABSTRACT
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 pKa(eff) values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKa(eff) values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)-WT1-4, Kf(ML) value of 7.5 × 10(12) 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 Kf(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.

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Speciation diagram of the Zn(II)–WT1-4 metal–ligand complex depicting the diimidazole–dithiolatezinc species (solid line), Zn(II)–WT1-4, the diimidiazole–dithiol zinc species (dashed line), Zn(II)–WT1-4–2H+, and the diimidazolium–dithiolspecies (dotted line), Zn(II)–WT1-4–4H+. The diagram was generated on the basis ofthe protonation behavior of the Zn(II)–WT1-4 complex in Figure 7.
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fig8: Speciation diagram of the Zn(II)–WT1-4 metal–ligand complex depicting the diimidazole–dithiolatezinc species (solid line), Zn(II)–WT1-4, the diimidiazole–dithiol zinc species (dashed line), Zn(II)–WT1-4–2H+, and the diimidazolium–dithiolspecies (dotted line), Zn(II)–WT1-4–4H+. The diagram was generated on the basis ofthe protonation behavior of the Zn(II)–WT1-4 complex in Figure 7.

Mentions: The pKaeff valuesof Zn(II)–WT1-4 were measured usingpotentiometric pH titrations followed by fluorescence spectroscopyto determine the appropriate Zn(II) proton competition equilbriummodel. Figure 7 shows that titration of 0.1M HCl into 15 μM Zn(II)–WT1-4 results in a decrease in tryptophan fluorescence. The pH titrationof Zn(II)–WT1-4 is best fit to anequilibrium model involving two distinct two-proton protonation events,a coupled two-proton event at a pKa1,2eff value of 2.6 and a cooperative two-proton event ata pKa3,4eff value of 5.2, asshown in Scheme 1. These are assigned to theprotonation of the Zn(II)-bound His residues (2.6) and Cys (5.2) residues(Table 1), as shown in eqs 17 and 18.1718The measurement of these effectivepKaeff values is critical toproviding the correct Zn(II)–H+ competition modelfor the equilibrium presented in Scheme 1 andthe speciation diagram for Zn(II)–WT1-4 shown in Figure 8. At pH values greater than 6.5, Zn(II)–WT1-4 is predominantly in the Cys2His2, cysteine thiolate/histidine imidazole, form, asshown in Figure 8. Between pH 3.0 and 5.0,the major species is Zn(II)–WT1-4 in the (CysH+)2His2, cysteine thiol/histidineimidazole, form. Lastly, below pH 2.5, the WT1-4 peptide exists mostly in the (CysH)2His2, cysteine thiol/histidine imidazolium, form without the metal bound.


Characterization of the Zn(II) binding properties of the human Wilms' tumor suppressor protein C-terminal zinc finger peptide.

Chan KL, Bakman I, Marts AR, Batir Y, Dowd TL, Tierney DL, Gibney BR - Inorg Chem (2014)

Speciation diagram of the Zn(II)–WT1-4 metal–ligand complex depicting the diimidazole–dithiolatezinc species (solid line), Zn(II)–WT1-4, the diimidiazole–dithiol zinc species (dashed line), Zn(II)–WT1-4–2H+, and the diimidazolium–dithiolspecies (dotted line), Zn(II)–WT1-4–4H+. The diagram was generated on the basis ofthe protonation behavior of the Zn(II)–WT1-4 complex in Figure 7.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4066921&req=5

fig8: Speciation diagram of the Zn(II)–WT1-4 metal–ligand complex depicting the diimidazole–dithiolatezinc species (solid line), Zn(II)–WT1-4, the diimidiazole–dithiol zinc species (dashed line), Zn(II)–WT1-4–2H+, and the diimidazolium–dithiolspecies (dotted line), Zn(II)–WT1-4–4H+. The diagram was generated on the basis ofthe protonation behavior of the Zn(II)–WT1-4 complex in Figure 7.
Mentions: The pKaeff valuesof Zn(II)–WT1-4 were measured usingpotentiometric pH titrations followed by fluorescence spectroscopyto determine the appropriate Zn(II) proton competition equilbriummodel. Figure 7 shows that titration of 0.1M HCl into 15 μM Zn(II)–WT1-4 results in a decrease in tryptophan fluorescence. The pH titrationof Zn(II)–WT1-4 is best fit to anequilibrium model involving two distinct two-proton protonation events,a coupled two-proton event at a pKa1,2eff value of 2.6 and a cooperative two-proton event ata pKa3,4eff value of 5.2, asshown in Scheme 1. These are assigned to theprotonation of the Zn(II)-bound His residues (2.6) and Cys (5.2) residues(Table 1), as shown in eqs 17 and 18.1718The measurement of these effectivepKaeff values is critical toproviding the correct Zn(II)–H+ competition modelfor the equilibrium presented in Scheme 1 andthe speciation diagram for Zn(II)–WT1-4 shown in Figure 8. At pH values greater than 6.5, Zn(II)–WT1-4 is predominantly in the Cys2His2, cysteine thiolate/histidine imidazole, form, asshown in Figure 8. Between pH 3.0 and 5.0,the major species is Zn(II)–WT1-4 in the (CysH+)2His2, cysteine thiol/histidineimidazole, form. Lastly, below pH 2.5, the WT1-4 peptide exists mostly in the (CysH)2His2, cysteine thiol/histidine imidazolium, form without the metal bound.

Bottom Line: 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 Kf(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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Brooklyn College , 2900 Bedford Avenue, Brooklyn, New York 11210, United States.

ABSTRACT
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 pKa(eff) values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKa(eff) values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)-WT1-4, Kf(ML) value of 7.5 × 10(12) 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 Kf(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.

Show MeSH
Related in: MedlinePlus