Deducing the Energetic Cost of Protein Folding in Zinc Finger Proteins Using Designed Metallopeptides

Abstract

Zinc finger transcription factors represent the largest single class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys4, Cys3His1, or Cys2His2 sites results in metal-induced protein folding events required to achieve their proper structure for biological activity. The thermodynamic contribution of Zn(II) in each of these coordination spheres toward protein folding is poorly understood because of the coupled nature of the metal-ligand and protein-protein interactions. Using an unstructured peptide scaffold, GGG, we have employed fluorimetry, potentiometry, and calorimetry to determine the thermodynamics of Zn(II) binding to the Cys4, Cys3His1, and Cys2His2 ligand sets with minimal interference from protein folding effects. The data show that Zn(II) complexation is entropy driven and modulated by proton release. The formation constants for Zn(II)-GGG with a Cys4, Cys3His1, or Cys2His2 site are 5.6 x 10(16), 1.5 x 10(15), or 2.5 x 10(13) M(-1), respectively. Thus, the Zn(II)-Cys4, Zn(II)-Cys3His1, and Zn(II)-Cys2His2 interactions can provide up to 22.8, 20.7, and 18.3 kcal/mol, respectively, in driving force for protein stabilization, folding, and/or assembly at pH values above the ligand pKa values. While the contributions from the three coordination motifs differ by 4.5 kcal/mol in Zn(II) affinity at pH 9.0, they are equivalent at physiological pH, DeltaG = -16.8 kcal/mol or a Ka = 2.0 x 10(12) M(-1). Calorimetric data show that this is due to proton-based enthalpy-entropy compensation between the favorable entropic term from proton release and the unfavorable enthalpic term due to thiol deprotonation. Since protein folding effects have been minimized in the GGG scaffold, these peptides possess nearly the tightest Zn(II) affinities possible for their coordination motifs. The Zn(II) affinities in each coordination motif are compared between the GGG scaffold and natural zinc finger proteins to determine the free energy required to fold the latter. Several proteins have identical Zn(II) affinities to GGG. That is, little, if any, of their Zn(II) binding energy is required to fold the protein, whereas some have affinities weakened by up to 5.7 kcal/mol; i.e., the Zn(II) binding energy is being used to fold the protein.

Cooperative Metal Binding and Helical Folding in Model Peptides of Treble‐Clef Zinc Fingers

“…pH, buffer, direct titration or competition experiments). As pointed out by the studies of Mely et al [36] and Gibney et al, [12,14,30] as a consequence of the competition between metal and protons, the pH-dependence of the apparent metal binding constant must be investigated to fully describe the coordination properties (species formed and binding constants) of the cysteine-contaning peptides. The protonation state of cysteines in ZnA C H T U N G T R E N N U N G (Cys) 4 …”

“…For example, the cost of the folding of CCHH zinc fingers is still subject to debate in spite of a high number of studies. [13,14] Small molecular models of protein metal sites constitute a viable tool to gain insights into the metal/protein interactions. [15] Determining the energy contributions of these interactions is crucial to understand metal-induced protein folding and design de novo metalloproteins.…”

“…Moreover the shape of the d-d transition pattern of these CoA C H T U N G T R E N N U N G (Cys) 4 centres is extremely sensitive to their structure, as we showed for Hsp33 models. Figure S3 (in the Supporting Information) compares the visible spectrum of Co·L TC to that of the Co 2 + -loaded GATA protein, which possesses a treble-clef structural site, [25][26][27] and those of Co 2 + -loaded Hsp 33 [28] and CP-(CCCC) zinc finger [29] consensus peptide, which have different folds, and those of the unstructured GGG-Cys 4 [14] and IGA-Cys 4 [30] peptides. [31] This comparison shows unambiguously that Co 2 + binding to the proper folding of the treble-clef motif gives a unique signature that is matched by Co·L TC .…”

Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure

“…So the overall folding involves a delicate interplay of hydrophobic packing, hydrogen bonding, and metal binding (Li et al, 2007(Li et al, , 2008. In addition, if the coordination residues are located on flexible terminals or loops, as in BABZ2 and BABZ4, zinc binding-induced aggregation may occur, especially for the small protein like βαβ, so the entropy cost to fix the coordination residues at specific conformations should be considered, as zinc binding in proteins is an entropy-driven process (Reddi et al, 2007). It has been demonstrated that the primary interactions are enough to produce potentially useful zinc binding affinity (Wade et al, 1993), and this agrees with our design work.…”

Applications of polymer single nanochannels in biosensors

“…After Zn 2+ was added, the channel was activated showing an obvious increase in ion conductance. Zinc finger proteins shrank to finger-like conformations, thus enlarged the effective pore size [58]. Appropriate opening size of nanochannel is necessary for a successful zinc biosensor.…”