Zinc finger proteins function in the regulation of gene expression and DNA metabolism. Although the structural diversity of zinc coordination complexes continues to grow, all zinc finger proteins incorporate one or more structural Zn(II) ions as a means to stabilize functionally important region(s) of the molecule. The long term goal of this work is to arrive at an understanding as to how nature obtains an appropriate degree of thermodynamic and kinetic stability in these zinc complexes. There are two aims in this proposal. First, two model systems, phage T4 gene 32 protein and a viral nucleocapsid protein have been selected to begin to define the physical principles which optimize the coordination and stability of zinc finger proteins. A systematic metal-site redesign or metal-ligand substitution strategy will be employed to determine the origin of the stability of the metal chelate and evaluate to what extent alternative coordination geometries and liganding sets are accommodated in each protein, ie., to probe the """"""""plasticity"""""""" of the domain. Metal ligand sets and their thermodynamic stabilities of wild-type and mutant coordination complexes will be defined by optical absorption spectroscopy of the Co(II)-substituted proteins and by (113)Cd(II) substitution and NMR spectroscopy.
The second aim addresses a proposed regulatory function that zinc coordination complexes of perhaps lower thermodynamic stability play in the transcriptional activation of metallothionein gene expression induced by heavy metals. The human metallothionein-I promoter contains multiple metal responsive elements, MREs, which specifically bind the Cys(2)-His(2) zinc finger protein, MTF-1. MTF-1 has been proposed to play a metalloregulatory role by directly sensing changes in intracellular zinc concentration and inducing MT-I expression; Sp1, also a Cys(2)-His(2) zinc finger protein, is not similarly regulated by zinc. To test this hypothesis and gain mechanistic insight into zinc regulated gene expression, the apparent overall and stepwise equilibrium association constants for Zn(II) and other metals with recombinant Sp1 and MTF-1 will be compared using optical absorption spectroscopy and metal competition methods. A rigorous fluorescence-based specific DNA- binding assay has been developed to determine the quantitative thermodynamic linkage relationships between the binding of Zn(II), a positive allosteric activator of zinc finger protein activity, and sequence-specific DNA binding by Sp1 vs. human MTF-1 using purified macromolecular components.
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