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Salt effects and DNA-binding specificity of NZF-1, a CCHC zinc binding protein
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Journal of Inorganic Biochemistry 96 (2003)
Salt Effects and DNA-binding Specificity of NZF-1, a CCHC Zinc Binding Protein. Alisa M Davis, Johns Hopkins University School of Medicine, UnitedStates Holly J Berkovits, Johns Hopkins Universi@ School of Medicine, UnitedStates Barbara T Amann, Johns Hopkins University School ofMedicine, United States Jeremy M Berg, Johns Hopkins University School of Medicine, United States The first type of zinc-binding domain described in transcription factors contains a Cys,His, motif. Recently, new families of transcription factors characterized by a CCHC zinc binding motif have been identified. Here we describe further characterization of a two-domain fragment, NZFl-2D, from the transcription factor, Neural Zinc Finger Factor 1 (NZF-1). NZF-1 contains six conserved CCHHC zinc-binding motifs and is involved in the development of the nervous system in eukaryotes by binding preferentially to the beta-retinoic acid receptor (P-RARE) DNA sequence. Gel-shifi studies identified a seven-oligonucleotide tandem repeat within the P-RARE DNA sequence that appears to be essential for NZF- 1 binding, with an estimated K, of 1Ow6 M. We present here the quantitative determination of this binding interaction using fluorescence anisotropy. In addition, we have investigated the effect of sodium chloride concentration on the specificity of P-RARE -NZFl-2D binding and have determined that the binding interaction is significantly compromised at salt concentrations less than and greater than 0.1 M. These data will be useful in continued structural studies of the P-RARE - NZFl-2D complex,
Characterization of the diiron(II1) site in stearoyl-acp desaturase through EPR and ENDOR of the protein radiolytically reduced at 77k: effect of substrate. Roman Davvdov, Department of Chemistry, Northwestern University, United States Brian M Hoffman, Department of Chemistry, Northwestern University, United States Behnaz Behrouzian, Department of Biology Brookhaven National Laboratory, United States John Shanklin, Department of Biology, Brookhaven National Laboratory, United States The diiron center in stearoyl-ACP desaturase (DS) from castor plant catalyzes the dioxygen and NADPH-dependent introduction of a cis-double bond between C9 and Cl0 of stearoyl-ACP. Radiolytic reduction of diferric DS at 77K produces an EPR detectable mixed-valence center trapped in the conformation of the diferric precursor that is a sensitive EPIVENDOR probe for structural study of the diamagnetic diiron(II1) state. Cryoreduced DS shows two distinct EPR signals suggesting the presence oftwo conformers with the p-oxo(major) and p-hydroxo(minor) bridged diiron centers. ENDOR studies show that in dominant conformer each Fe(II1) coordinates a histidine and a water along with other ligands. Addition of stoichiometric amounts of stearoyl-ACP results in pronounced changes in the EPR and ENDOR spectra of cryoreduced DS. EPR spectra of cryoreduced DS-substrate complex disclose two distinct conformational states for DS. ENDOR studies show that in the major conformer each iron of the diferric cluster coordinates a p-0x0 ligand, water and histidine. In the minor conformer, one of the irons of the active site loses the terminal water ligand. The p-0x0 bridge in the major cryoreduced DS species is protonated on annealing to 230K, while no protonation of the p-0x0 bridge is observed upon annealing cryoreduced DSsubstrate complex. The mixed-valence states of DS disappear at T>24OK likely via disproportionation. The alterations in the diferric site of DS induced by the substrate are suggested to be mediated by conformational changes in the polypeptide chain produced by substrate binding. These structural alterations may provide DS with an additional mechanism for tuning the redox potential of the active site.
Journal of Inorganic Biochemistry 96 (2003)
Salt Effects and DNA-binding Specificity of NZF-1, a CCHC Zinc Binding Protein. Alisa M Davis, Johns Hopkins University School of Medicine, UnitedStates Holly J Berkovits, Johns Hopkins Universi@ School of Medicine, UnitedStates Barbara T Amann, Johns Hopkins University School ofMedicine, United States Jeremy M Berg, Johns Hopkins University School of Medicine, United States The first type of zinc-binding domain described in transcription factors contains a Cys,His, motif. Recently, new families of transcription factors characterized by a CCHC zinc binding motif have been identified. Here we describe further characterization of a two-domain fragment, NZFl-2D, from the transcription factor, Neural Zinc Finger Factor 1 (NZF-1). NZF-1 contains six conserved CCHHC zinc-binding motifs and is involved in the development of the nervous system in eukaryotes by binding preferentially to the beta-retinoic acid receptor (P-RARE) DNA sequence. Gel-shifi studies identified a seven-oligonucleotide tandem repeat within the P-RARE DNA sequence that appears to be essential for NZF- 1 binding, with an estimated K, of 1Ow6 M. We present here the quantitative determination of this binding interaction using fluorescence anisotropy. In addition, we have investigated the effect of sodium chloride concentration on the specificity of P-RARE -NZFl-2D binding and have determined that the binding interaction is significantly compromised at salt concentrations less than and greater than 0.1 M. These data will be useful in continued structural studies of the P-RARE - NZFl-2D complex,
Characterization of the diiron(II1) site in stearoyl-acp desaturase through EPR and ENDOR of the protein radiolytically reduced at 77k: effect of substrate. Roman Davvdov, Department of Chemistry, Northwestern University, United States Brian M Hoffman, Department of Chemistry, Northwestern University, United States Behnaz Behrouzian, Department of Biology Brookhaven National Laboratory, United States John Shanklin, Department of Biology, Brookhaven National Laboratory, United States The diiron center in stearoyl-ACP desaturase (DS) from castor plant catalyzes the dioxygen and NADPH-dependent introduction of a cis-double bond between C9 and Cl0 of stearoyl-ACP. Radiolytic reduction of diferric DS at 77K produces an EPR detectable mixed-valence center trapped in the conformation of the diferric precursor that is a sensitive EPIVENDOR probe for structural study of the diamagnetic diiron(II1) state. Cryoreduced DS shows two distinct EPR signals suggesting the presence oftwo conformers with the p-oxo(major) and p-hydroxo(minor) bridged diiron centers. ENDOR studies show that in dominant conformer each Fe(II1) coordinates a histidine and a water along with other ligands. Addition of stoichiometric amounts of stearoyl-ACP results in pronounced changes in the EPR and ENDOR spectra of cryoreduced DS. EPR spectra of cryoreduced DS-substrate complex disclose two distinct conformational states for DS. ENDOR studies show that in the major conformer each iron of the diferric cluster coordinates a p-0x0 ligand, water and histidine. In the minor conformer, one of the irons of the active site loses the terminal water ligand. The p-0x0 bridge in the major cryoreduced DS species is protonated on annealing to 230K, while no protonation of the p-0x0 bridge is observed upon annealing cryoreduced DSsubstrate complex. The mixed-valence states of DS disappear at T>24OK likely via disproportionation. The alterations in the diferric site of DS induced by the substrate are suggested to be mediated by conformational changes in the polypeptide chain produced by substrate binding. These structural alterations may provide DS with an additional mechanism for tuning the redox potential of the active site.