9 research outputs found

    Unique Spectroscopic Properties of the H‑Cluster in a Putative Sensory [FeFe] Hydrogenase

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    Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H<sub>2</sub> level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from <i>Thermotoga maritima</i> (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the H<sub>ox</sub> state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the H<sub>ox</sub> state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster

    DNA binding studies of INI1 DBD.

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    <p>(A) Coomassie blue stained SDS-PAGE (left panel) and Western blot analysis (right panel) of recombinant, purified INI1 DBD using α-His antibodies as probes. (B) Agarose gel retardation assay (AGRA) of INI1 DBD using 100 ng of pET28a as substrate and indicated amount of polypeptide; RC: relaxed circular DNA, CCC: covalently closed circular DNA. (C) Ni-NTA pull-down assay of INI1 DBD:U5 HIV-1 LTR complex (lane a) and of DNA alone (lane b). The precipitated complex was analyzed by western blot using α-His antibodies as probes to detect INI1 DBD and the co-precipitated DNA was analyzed by ethidium bromide (EtBr) staining following 10% urea-PAGE. The loading controls are shown. (D) Atomic force microscopy (AFM) images of pNEB206A DNA alone (left panel) and in complex with INI1 DBD (right panel). Regions of DNA coated with protein (arrows) and free DNA (arrowhead) is shown.</p

    Sedimentation equilibrium analysis of INI1 DBD and INI1:DNA complex.

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    <p>(A) Representative sedimentation equilibrium (SE) profiles of 10 µM (A) and 60 µM (B) of INI1 DBD generated from data collected at 280 nm. Representative sedimentation equilibrium (SE) profiles of a reaction mixture of 10 µM INI1 DBD and 10 µM U5 HIV-1 LTR DNA diluted 1∶7.5 x with buffer (C) and a reaction mixture of 60 µM INI1 DBD and 60 µM U5 HIV-1 LTR DNA diluted 1∶40 x with buffer (D) generated from data collected at 260 nm. Lower panels: Radial distribution of the concentration of INI1 DBD (A and B) and DNA (C and D) both free and in complex with INI1 DBD at sedimentation equilibrium. The solid line represents best fit. Upper panels: Distributions of the residuals around a zero mean.</p

    Phylogenetic conservation of the region corresponding to amino acids 105-183 of INI1.

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    <p>(A) Scheme showing presence and absence of different domains in INI1 from yeast to humans. (B) Neighbor-joining tree using MEGA 2.0 software showing conservation of the region corresponding to amino acids 105-183 of INI1. Percentage identity of the region in different species is shown in parenthesis.</p

    Determination of stoichiometries of INI1 DBD binding to U5 HIV-1 LTR DNA from analytical ultracentrifugation studies.

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    a<p>Calculated from amino acid or nucleotide composition, <i><sup>b</sup></i>Based upon sedimentation equilibrium data, ± = SD (standard deviation).</p

    Model showing different modes of INI1 DNA binding.

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    <p>The INI1 DBD undergoes concentration dependent multimerization. Two molecules of monomeric DBD binds to one molecule of U5 HIV-1 LTR DNA whereas one molecule of dimeric DBD binds to two molecules of U5 HIV-1 LTR DNA.</p

    Sequence conservation of the putative DNA binding domain (DBD) of vertebrate INI1/hSNF5, Drosophila SNR1 and <i>C.</i><i>elegans</i> SNF5.

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    <p>(A) Sequence comparison of the putative DNA binding domain of Drosophila SNR1 with the domains in vertebrates. Conserved residues are shown by (*) and partially conserved residues by (#). The HH and KKR motifs are shown with dash and broken dash, respectively. The conserved cysteine residues are shown by an arrow. The conserved lysine, arginine and histidine residues are shown with an arrowhead. (B) Sequence comparison of the putative DNA binding domain of <i>C. elegans</i> SNF5 with the human domain.</p

    Isothermal calorimetry.

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    <p>ITC of INI1 DBD binding to U5 HIV-1 LTR DNA using 10 µM INI1 DBD (monomer) and 55 µM U5 HIV-1 LTR DNA (A) and 30 µM INI1 DBD (dimer) and 325 µM U5 HIV-1 LTR DNA (B). Top panel: Raw data of heat associated with mixing of DNA and protein. Heat of dilution is shown in the inset (not to scale). Bottom panel: Heat associated with each injection is obtained by integration of the area under the peak as a function of time. Binding curve (fitted) obtained by subtracting heat of dilution from heat associated with mixing of protein and DNA. Data is representative of multiple experiments.</p
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