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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 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 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.

Metal response element (MRE) binding transcription factor-1 (MFT1) is a six Cys2His2 zinc finger-containing transcription factor required for basal and zinc-induced transcription of metallothionein genes. Metallothioneins protein plays an important role in metal ion regulation in eukaryotic cells.

The structure of a CCHHC Zinc-binding domain from neural zinc finger factor-1 (NZF-1) is obtained in solution though the use of NMR methods. Studies reveal that a two-domain protein fragment interacts with a half-site within a pseudosymmetric DNA binding site and the structure of the domain helps in identifying residues that play significant roles in DNA binding and for designing the future structure.

The use of engineered zinc finger domains and their use for the design of sequence-specific DNA binding proteins are presented. Key examples that reveal the approaches are discussed in detail.

Azurin binds copper(II) quite tightly and exchanges metal ions very slowly. Isothermal titration calorimetry has the potential to reveal key thermodynamic parameters including both the standard free energy and enthalpy of metal binding. The response after each injection of apoazurin shows an initial exothermic phase followed by an endothermic phase.

Research shows that the zinc-binding domain of tristetraprolin D1 binds strongly the oligonucleotide with a sequence of UUUAUUU with a dissociation constant at the low micromolar range. The binding is selecive for the oligonucleotide sequence and RNA is preferred over DNA bearing the same sequence.

The peroxisomal lumen proteins contain a C-teminal tripeptide peroxisomal targeting signal-1 (PTS1), which is recognized the tetratricopeptide repeat domains of PEX5. Results show that PTS1 variants bind to the PEX5 fragment and that a binding energy threshold perhaps determines the PTS1 sequence functionality.

Tandem arrays of Cys2His2 zinc finger domains have the most effective framework for the development of novel DNA-binding proteins. A structural study of Cys2His2 zinc finger proteins in complex with DNA has revealed a conserved betabetaalpha architecture stabilized by the coordination of a zinc atom by two cysteines and two histidines.

A zinc (Zn) consensus peptide (CP), whose fluorescence emission spectrum changes due to protein folding induced by Zn binding, is an efficient Zn probe. The CP contains the fluorescein (F) and lissamine (L) fluorescent dyes that are separated in the unfolded protein. Protein folding brings the F and L together, and this changes the intramolecular energy transfer and the emission spectrum. CP-L-F's spectrum has two absorption peaks at 495 and 578 nm that correspond to conjugated F and L respectively. The fluorescent dyes have no adverse effect on the metal binding and folding properties of CP.

The racemic rubredoxin (RbDD) protein, commonly extracted from the bacterium Desulfovibrio desulfuricans, was successfully prepared from its enantiomeric forms. The protein exhibited high metal binding affinities that facilitated the formation of Co2+ and Fe2+ complexes. Circular dichroism measurements confirmed the presence of the racemate form of the protein in synthetically purified RbDD crystals.

Most proteins are targeted to the peroxisomal matrix by virtue of a peroxisomal targeting signal-i (PTS1), a short carboxy-terminal sequence specifically recognized by the PTS1 receptor Pex5p. We had previously developed a model that allowed the estimation of the affinities of many PTS1 sequences within the human proteome for Pex5p that revealed a wide range of predicted affinities. We have now experimentally determined the affinities of the PTS1-containing peptides from 42 proteins from the human proteome for Pex5p and show that these range over 4 orders of magnitude. These affinities correlate reasonably well with the predicted values and are substantially more precise. In an attempt to provide a possible explanation for the wide range of PTS1 -Pex5p affinities, we compared these affinities with mRNA levels (as a proxy for rates of protein production) of the genes encoding these proteins in 79 human tissues and cell types. We note that high affinity PTS1 -Pex5p interactions tend to correspond to proteins encoded by genes expressed at relatively low levels, whereas lower affinity PTS1 -Pex5p interactions tend to correspond to proteins encoded by genes exhibiting higher levels and wider ranges of expression. Further analysis revealed that these relationships are consistent with the notion that a relatively uniform pool of protein-Pex5p complexes is maintained for appropriate peroxisome assembly. [ABSTRACT FROM AUTHOR]

Metal response element (MRE) binding transcription factor-1 (M'l'F1) is a six Cys2His2 zinc finger-containing transcription factor required for basal and zinc-induced transcription of metallothionein genes. The cobalt(II) and zinc(II) affinities of a protein fragment comprising the six zinc finger domains have been examined to reveal apparent dissociation constants (for the six domains collectively) of 0.5 ± 0.2 μM for cobalt(II) and 31 ± 14 μM for zinc(II). Two approaches have been used to determine the metal ion affinities of the individual domains. First, the six domains have been examined as single domain peptides revealing dissociation constants ranging from 0.3 to 1.7 μM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (Kd(CO(II)) 0.3-0.5 μM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (Kd(CO(II)) 1.6-1.7 μM). Second, we examined the affinity of each domain in the context of the six zinc finger domain protein by individually mutating one metal-binding His residue to Cys to allow independent monitoring of the cobalt(II) occupancy of each site. The affinity of each domain was higher in this context than as a single domain peptide with affinities (corrected for the effect of the mutation) ranging from 0.02 to 0.5 μM. The increase in affinity for the individual domains ranged from factors of 1.1 to 20. The order of affinities (from higher to lowest) was observed to be 4 > 2 ≈ 5 > 6 ≈ 3 ≈ 1. These results reveal that none of the Cys2His2 zinc finger domains of MTF1 have dramatically low metal ion affinities, certainly none low enough to respond to changes in free zinc ion concentrations in the micromolar range. Nonetheless, the metal ion affinities of some domains do differ by a fadtor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains. [ABSTRACT FROM AUTHOR]

Focuses on research that deals with zinc finger protein and DNA, RNA and DNA-RNA hybrids in relation to protein binding. Details on research; Experimental procedures; Results and discussion.

Polycomb group (PcG) proteins maintain the silent state of developmentally important genes. Recent evidence indicates that noncoding RNAs also play an important role in targeting PcG proteins to chromatin and PcG-mediated chromatin organization, although the molecular basis for how PcG and RNA function in concert remains unclear. The Phe-Cys-Ser (FCS) domain, named for three consecutive residues conserved in this domain, is a 30-40-residue Zn(2+) binding motif found in a number of PcG proteins. The FCS domain has been shown to bind RNA in a non-sequence specific manner, but how it does so is not known. Here, we present the three-dimensional structure of the FCS domain from human Polyhomeotic homologue 1 (HPH1, also known as PHC1) determined using multidimensional nuclear magnetic resonance methods. Chemical shift perturbations upon addition of RNA and DNA resulted in the identification of Lys 816 as a potentially important residue required for nucleic acid binding. The role played by this residue in Polyhomeotic function was demonstrated in a transcription assay conducted in Drosophila S2 cells. Mutation of the Arg residue to Ala in the Drosophila Polyhomeotic (Ph) protein, which is equivalent to Lys 816 in HPH1, was unable to repress transcription of a reporter gene to the level of wild-type Ph. These results suggest that direct interaction between the Ph FCS domain and nucleic acids is required for Ph-mediated repression.

The most exciting and vibrant areas of research often lie at the interfaces between disciplines that have traditionally been separate. In the case of the chemical and biological sciences, such interfaces have been fruitfully explored for more than a century. The fields of pharmacology, biochemistry, and biophysical chemistry are relatively mature, yet they are still quite active and full of challenging problems and opportunities for new discoveries. These fields are integral to biomedical research and have had a tremendous impact on human health.

Nup475 (also known as tristetraprolin and TIS11) includes two zinc-binding domains of the form Cys-X8-Cys-X5-Cys-X3-His. These domains are required for rapid degradation of tumor necrosis factor (TNF) and other mRNAs through the interaction with AU-rich elements in their 3'-untranslated regions. The three-dimensional solution structure of the first domain was determined by multidimensional nuclear magnetic resonance spectroscopy, revealing a novel fold around a central zinc ion. The core structure is disk-like with a diameter of approximately 25 A and a width of approximately 12 A. This structure provides a basis for evaluating the role of individual residues for structural stability and for nucleic acid binding.