title: Evaluation of the Intrinsic Zn(II) Affinity of a Cys3His1 Site in the Absence of Protein Folding Effects.
abstract: Zinc finger transcription factors are the largest class of metalloproteins in the human genome. Binding of Zn(II) to their canonical Cys2His2, Cys3His1, or Cys4 sites results in metal-induced protein folding events required to achieve their biologically active structures. However, the coupled nature of metal binding and protein folding obscures the individual free energy contributions of each process toward overall zinc finger stabilization. Herein, we separate the energetic contributions of metal-ligand interactions from those of protein-protein interactions using a natural protein scaffold that retains essentially identical structures with and without Zn(II) bound, the 59 amino acid zinc binding domain of human transcription factor IIB (ZBD-TFIIB). The formation constant of Zn(II)-ZBD-TFIIB, which contains a single Cys3His1 site, was determined to be 1.5 × 10(15) M(-1) via fluorimetry and isothermal titration calorimetry. Isothermal titration calorimetry showed that Zn(II) binding is entropically favored at pH 5.5, 7.0, and 8.0 and enthalpically favored at pH 8.0 but slightly enthalpically disfavored at pH 5.5 and 7.0. The conditional dissociation constants of Zn(II)-ZBD-TFIIB and natural Cys3His1 zinc finger proteins were compared to determine the free energy cost of protein folding in the latter. Our analysis reveals that the energetic cost to fold zinc finger proteins is minimal relative to the contribution of Zn(II) binding and suggests that the true role of Zn(II) binding may be to modulate protein dynamics and/or kinetically template the protein folding process.
authors.forename: Amit R
authors.initials: AR
authors.lastname: Reddi
authors.affiliation: ‡Department of Chemistry, Brooklyn College, 2900 Bedford Avenue, Brooklyn, New York 11210, United States.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Pawlowska
authors.affiliation: ‡Department of Chemistry, Brooklyn College, 2900 Bedford Avenue, Brooklyn, New York 11210, United States.|§Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, New York 10016, United States.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Gibney
mesh_terms: D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D035581:Transcription Factor TFIIB / Q000737:chemistry* / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
chemical_list: D035581:Transcription Factor TFIIB; D015032:Zinc