7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV‐2) associated protein gp125. Fab fragments of 7C8 ...effectively neutralize HIV‐2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity‐determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size‐exclusion chromatography. Hanging‐drop vapour‐diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris–HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3221, with unit‐cell parameters a = b = 100.1, c = 196.8 Å, and diffracted to 2.7 Å resolution.
The crystal structures of the three-component toluene 2, 3-dioxygenase system provide a model for electron transfer among bacterial Rieske non-heme iron dioxygenases. Bacterial Rieske non-heme iron ...oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2, 3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske 2Fe–2S cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a 2Fe–2S cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.
Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule ...fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.
Pseudomonas putida F1 can grow with toluene as its sole source of carbon and energy. The initial reaction of the degradation of toluene is catalyzed by a three-component toluene dioxygenase enzyme ...system consisting of a reductase (Reductase{sub TOL}), a ferredoxin (Ferredoxin{sub TOL}) and a Rieske non-heme iron dioxygenase (Oxygenase{sub TOL}). The three components and the apoenzyme of the dioxygenase (apo-Oxygenase{sub TOL}) were overexpressed, purified and crystallized. Reductase{sub TOL} diffracts to 1.8 {angstrom} and belongs to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 77.1, c = 156.3 {angstrom}. Ferredoxin{sub TOL} diffracts to 1.2 {angstrom} and belongs to space group P2{sub 1}, with unit-cell parameters a = 30.5, b = 52.0, c = 30.95 {angstrom}, {beta} = 113.7{sup o}. Apo-Oxygenase{sub TOL} and Oxygenase{sub TOL} diffract to 3.2 {angstrom} and belong to space group P4{sub 3}32, with unit-cell parameters a = 235.9 {angstrom} and a = 234.5 {angstrom}, respectively.
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