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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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Kinetics of Zn(II) removal from 22 μM Zn(II)–WT1-4 by 100 μM EDTA buffered at pH 6.65 (20 mM MES,100 mM KCl) followed by the decrease in fluorescence emission intensityat 355 nm. The fluorescence emission intensity drops from an initialvalue of 145.67 au to 78.45 au in 15 min. Under similar conditions,Sénèque and Latour15 measuredan equilibration time for Zn(II)–CP1-CCHH of 1600 min.
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fig5: Kinetics of Zn(II) removal from 22 μM Zn(II)–WT1-4 by 100 μM EDTA buffered at pH 6.65 (20 mM MES,100 mM KCl) followed by the decrease in fluorescence emission intensityat 355 nm. The fluorescence emission intensity drops from an initialvalue of 145.67 au to 78.45 au in 15 min. Under similar conditions,Sénèque and Latour15 measuredan equilibration time for Zn(II)–CP1-CCHH of 1600 min.

Mentions: Figure 3 shows the direct titrationof Zn(II) into 22 μM WT1-4 at pH 5.25(20 mM MES, 100 mM KCl). The equilibrium binding isotherm shown inFigure 3 is fit to a 1:1 metal–peptidebinding model, eq 3, and demonstrates a conditionaldissociation constant value of 22 μM at pH 5.25. At pH valuesabove 6.0, competition titrations with the chelators HEDTA and EGTAwere performed to obtain accurate conditional dissociation constantvalues for Zn(II)–WT1-4. The formationconstants for Zn(II)–HEDTA and Zn(II)–EGTA and theirrespective pKa values were used to calculatetheir conditional dissociation constants between pH 6.0 and 9.0. Figure 4 shows a typical competition titration of Zn(II)into a buffered pH 9.0 (20 mM Tris, 100 mM KCl) solution containing22 μM WT1-4 and 110 μM HEDTA.The equilibrium binding isotherm shown in Figure 4 is fit to a competition constant of 3.31. Because the Kd of Zn(II)–HEDTA is 2.01 × 10–14 M at pH 9.0, the conditional dissociation constantof Zn(II)–WT1-4 is 6.6 × 10–14 M at pH 9.0. Kinetic experiments were performedto ensure that samples had reached equilibrium. Figure 5 shows the change in fluorescence intensity as a functionof time upon addition of 5 equiv of EDTA to 22 μM Zn(II)–WT1-4 in 20 mM MES, 100 mM KCl, at pH 6.65. Theinitial intensity, 145.67 au, decreases to 78.45 au in 15 min andremains constant for the following 45 min. Kinetic experiments fromdirect and competition titrations shows that all samples were equilibratedwithin 15 min, so measurements were taken 30 min after Zn(II) additionto ensure equilibration.


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)

Kinetics of Zn(II) removal from 22 μM Zn(II)–WT1-4 by 100 μM EDTA buffered at pH 6.65 (20 mM MES,100 mM KCl) followed by the decrease in fluorescence emission intensityat 355 nm. The fluorescence emission intensity drops from an initialvalue of 145.67 au to 78.45 au in 15 min. Under similar conditions,Sénèque and Latour15 measuredan equilibration time for Zn(II)–CP1-CCHH of 1600 min.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Kinetics of Zn(II) removal from 22 μM Zn(II)–WT1-4 by 100 μM EDTA buffered at pH 6.65 (20 mM MES,100 mM KCl) followed by the decrease in fluorescence emission intensityat 355 nm. The fluorescence emission intensity drops from an initialvalue of 145.67 au to 78.45 au in 15 min. Under similar conditions,Sénèque and Latour15 measuredan equilibration time for Zn(II)–CP1-CCHH of 1600 min.
Mentions: Figure 3 shows the direct titrationof Zn(II) into 22 μM WT1-4 at pH 5.25(20 mM MES, 100 mM KCl). The equilibrium binding isotherm shown inFigure 3 is fit to a 1:1 metal–peptidebinding model, eq 3, and demonstrates a conditionaldissociation constant value of 22 μM at pH 5.25. At pH valuesabove 6.0, competition titrations with the chelators HEDTA and EGTAwere performed to obtain accurate conditional dissociation constantvalues for Zn(II)–WT1-4. The formationconstants for Zn(II)–HEDTA and Zn(II)–EGTA and theirrespective pKa values were used to calculatetheir conditional dissociation constants between pH 6.0 and 9.0. Figure 4 shows a typical competition titration of Zn(II)into a buffered pH 9.0 (20 mM Tris, 100 mM KCl) solution containing22 μM WT1-4 and 110 μM HEDTA.The equilibrium binding isotherm shown in Figure 4 is fit to a competition constant of 3.31. Because the Kd of Zn(II)–HEDTA is 2.01 × 10–14 M at pH 9.0, the conditional dissociation constantof Zn(II)–WT1-4 is 6.6 × 10–14 M at pH 9.0. Kinetic experiments were performedto ensure that samples had reached equilibrium. Figure 5 shows the change in fluorescence intensity as a functionof time upon addition of 5 equiv of EDTA to 22 μM Zn(II)–WT1-4 in 20 mM MES, 100 mM KCl, at pH 6.65. Theinitial intensity, 145.67 au, decreases to 78.45 au in 15 min andremains constant for the following 45 min. Kinetic experiments fromdirect and competition titrations shows that all samples were equilibratedwithin 15 min, so measurements were taken 30 min after Zn(II) additionto ensure equilibration.

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