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[Cys.sub.2][His.sub.2] zinc finger proteins make up the largest class of transcription factors encoded in the genomes of higher eukaryotes. Recent studies of the Ikaros transcription factor demonstrated that this zinc finger protein undergoes cell cycle-dependent changes in association with DNA that seem to be due to phosphorylation of Thr or Ser residues in the linker regions connecting adjacent zinc finger domains. The high degree of conservation of this linker sequence within the [Cys.sub.2][His.sub.2] superfamily suggested a common mechanism for the cell cycle-dependent modulation of DNA-binding affinity throughout this large class of transcription factors. The effects of linker phosphorylation on DNA-binding affinity were investigated through a direct comparison of the DNA-binding properties of four synthetic zinc finger proteins produced by native chemical ligation. The four proteins, comprising three zinc finger domains joined by two consensus Thr-Gly-Glu-Lys-Pro linkers, correspond to all four possible combinations of linker Thr phosphorylation states. Fluorescence-based DNA-binding studies of specific DNA-binding site revealed that phosphorylation of a single linker reduced binding affinity [approximately equal to] 40-fold, whereas phosphorylation of both linkers reduced binding affinity 130-fold. These result with purified components demonstrate that linker phosphorylation does, indeed, produce a significant reduction in DNA-binding affinity and support a model wherein a single cell cycle-dependent Ser/Thr kinase could simultaneously inactivate a large number of zinc finger transcription factors.

Research has been conducted on tristetraprolin Nup475, zinc-binding domains. The three-dimensional solution structure of one of the domains has been investigated via the use of multidimensional nuclear magnetic resonance spectroscopy, and the results reveal a fold around the central zinc ion.

Research has been conducted on zinc finger peptide found to be unstrucrured in the absence of bound metal ions. The study of peptide' s metal binding thermodynamics carried out via the use of isothermal titration calorimetry is described.

The article presents the identification of a proteins family containing zinc-binding domains. The synthesis of a peptide corresponding to the putative zink-binding domain has been carried out.

Characterization of the two-domain fragment of NZF-1 has been performed. Cobalt(II) and zinc(II) binding properties of the metal binding domains have been investigated and zinc-induced protein folding has been observed.

The role of zinc ions in the activation of cysteine toward nucleophilic attack and the ability of the protein structure to regulate the reactivity of specific cysteine residues when more than one is bound to the same zinc ion were investigated. In addition, the effect of alkylation of cysteine on metal binding and stability of the peptide structure was examined. Experimental results showed that methylation affects the structure of the peptide-zinc complex in the last turn of the helix without disrupting the rest of the structure.

An analysis of the sequential metal binding by the RING finger domain of BRCA 1, an ovarian cancer susceptibilitty gene, was conducted using absorption spectroscopy. Results revealed that BRCA 1 RING finger peptide is structured at pH 7 without the need for metal ions, but with structural changes occurring after the metal binding. Furthermore, the interwoven structure suggested that metal binding occurred in an all-or-none method.

Rapidly exchanging (greater than 1.0s-super1) backbone amide protons exchange rates with solvent water in staphylococcal nuclease (SN) were measured at pH 6.03-7.03 with a 2D heteronuclear water exchange filter sequence. It was found that non-hydrogen-bonding and exposed residues had protection factors less than 15 for non-hydrophobic residues. Protection factors for buried and non-hydrogen-bonded protons were found to vary, indicating kinetically labile hydrogen bonds and solvent exposure by structural fluctuations.

The electrostatic interactions across the beta-sheet in the context of the CP-1 zinc finger peptide was analyzed during coupled peptide folding/metal binding reactions. Analysis of the electrostatic interactions in the model beta-sheet indicated the presence of modest free energies associated with potential ion pairs. The formation of the salt bridge in the beta-sheet was also characterized by the partial desolvation of the charged residues. Furthermore, the charged residues interact at a distance without the costs of desolvation and substantial conformational restriction.

Zinc finger proteins attach to DNA, RNA and DNA-RNA combinations. The DNA molecules of the resulting zinc finger protein-DNA complexes were observed to unwind. Conformational changes of DNA on zinc finger protein binding influenced protein-protein interactions which are essential in genetic transcription and zinc finger protein functions. The human transcriptor factor Sp1 unwound DNA upon protein binding. Similarly, designed zinc finger proteins unwound DNA structures upon binding.

The results of NMR studies of a paramagnetic ion Co(II) substituted zinc finger peptide are discussed. These determined the orientation and anisotropy of the magnetic susceptibility tensor. Two methods were used. The first method involved the assignment of resonances for the Co(II) followed by sensor refinement. The second method used an integrated spectrum and yielded a preliminary tensor that provided initial predictions for the Co(II) complex shifts.

The thermodynamic beta-sheet propensities of 20 common amino acids are measured using a zinc-finger host peptide. The amino acids were substituted into a guest site of the host system which were unfolded without bound metal. The propensities of each substance was determined using a competitive cobalt(II)-binding assay. The resulting scale indicated the thermodynamic origin of beta-sheet propensities.

The metal binding properties of the variants of CP-1(His24 to Cys) and CP-1(His20 to Cys, His24 to Cys) with Co2+, Zn2+ and Cd2+ were investigated. Competition experiments were performed to determine the ratio of the metal-peptide complex dissociation constants and the relative specificities of two peptides for different metal ions. Ligand field stabilization energy changes, ionic radius and polarizability were observed to affect metal ion binding specificity.

A polyanine 26- amino acid peptide characteristic of the TFIIIA-type zinc finger domains has been synthesized and characterized using its metal binding and structural properties. Results show that the peptide folds in the presence of appropriate metal ions to form a complex that has a three-dimensional structure similar to zinc finger domains with more natural sequences. The sequences form a 2:1 peptide/metal complex in which the cysteinate ligands are coordinated to the metal ion but not to histidine.

A pulsed field gradient NMR technique selectively identifies exchangeable proton signals. A frequency-selective gradient phase encoding of water protons facilitates the rapid saturation of water in this water-exchange filter (WEX filter). The WEX filter enables the determination of exchange rates, the resolution of overlapping exchangeable peaks and avoids sensitivity losses. This method is successfully applied for a sample of 2mM ubiquitin in 90/10 H2O/D2O, and determines the rate of exchange for a side-chain protein in the unfolded consensus zinc finger peptide CP1.

Initial studies were performed to examine the potential use of solution structures of nucleocapsid (NC)-like CCHC zinc-binding domains as modules to construct single-stranded nucleic acid binding peptides. The results provide evidence for the principle potential use of the NC-like CCHC zinc-binding domains as a basis for designing peptides that bind specific single-stranded nucleic acid sequences.

The affinities of peroxisomal targeting signal-1 (PTS1)-containing peptides are determined from 42 proteins from the human proteome for Pex5p.The high affinity PTS-1-Pex5p interactions have corresponded to proteins encoded by genes expressed at low levels, whereas lower affinity PTS1-Pex5p interactions have corresponded to proteins encoded by genes displaying higher levels and wider ranges of expression.

The ability of the zinc finger peptide to act as a ligand for other metals was investigated spectrophotometrically using the peptide CP-1. The peptide can bind Ni2+, Cu2+ and Fe2+ to produce complexes with spectroscopic properties consistent with distorted tetrahedral coordination by the cysteinate and histidine residues. The Cu2+ ion oxidizes the peptides into disulfide-linked species. The Ni2+ complexes are shown by spectra to be paramagnetic with distorted tetrahedral configurations. The specific reactivation of TFIIIA by zinc is caused by metal ion affinity.

A combination of size exclusion chromatography and other techniques are employed to explain the mechanisms associated with the homodimerization and heterodimerization of minimal zinc(II)-binding-domain peptides of T-Cell proteins CD4, CD8[alpha], and Lck. The various biological implications of the metal-bridged homodimers in T-cells are also discussed.

Several studies are conducted to explain the structure, as well as the function of the sterol carrier protein-2 (SCP-2) and the 20-amino acid N-terminal presequence. The presequence is shown to be extremely significant in regulating the structure of the protein.

The studies reveal that cobalt(II) affinities in the micromolar range could occur even for peptides that do not correspond to natural zinc-binding domains. The degree of distinction between authentic metal-binding domains and the corresponding linker-spanning peptides may be modest, at least for single domain peptide models.

Zinc (II) and cobalt (II) binding to a series of zinc finger peptides with different charged residue pairs across from one another in a beta sheet were examined. The importance of enthropy-enthalpy compensation in these relatively weak interactions is highlighted.