Cys(2)His(2) zinc finger proteins (ZFPs) adopt a highly conserved betabetaalpha-fold, which is stabilized by tetrahedral coordination of a central zinc ion by two cysteine and two histidine residues. Although the function of most zinc fingers is sequence-specific DNA binding, other roles such as zinc sensing have been identified. Here, methods are described to produce micromole quantities of zinc-sensing zinc finger domains from the Zap1 (two zinc fingers) and MTF-1 (six zinc fingers) transcription factors. Procedures are outlined to isolate recombinant zinc finger proteins from a bacterial expression system that generates both soluble intracellular (Zap1) and insoluble inclusion body (MTF-1) forms. Isolated proteins are reduced and subsequently HPLC purified at low pH and lyophilized, which generates proteins that are ideal for zinc-binding studies or metal substitution studies and stable during long-term storage. NMR and calorimetric methods are described to measure relative and individual zinc ion affinities in multi-fingered proteins, which is an essential step in unraveling the zinc-sensing mechanism of MTF-1 and Zap1.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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