Computational protocol: Molecular logic of the Zur-regulated zinc deprivation response in Bacillus subtilis

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[…] Modelled three-dimensional structure of BsuZur dimer was constructed after sequence alignment of the structure of the closely related Zur protein of S. coelicolor (PDB ID 3MWM) and built using PROMOD-II and MODELLER (http://swissmodel.expasy.org). The final 3D structure model was represented in the cartoon using PyMOL viewer program (2008. Open free version of DeLano Scientific). [...] Wild-type, C84S, C95S and H124A mutant Zur proteins were purified from E. coli BL21 (DE3) cells containing pET3a-based recombinant plasmid. The coding sequence for WT BsZur was cloned into pET3a (Novagen) between the NdeI and BamHI restriction sites. For BsZur mutants, plasmids were constructed using quick change site-directed mutagenesis and transformed into E. coli strain BL21 (DE3/pLysS). For the purification of Zur, an overnight culture from a single colony was used to inoculate 1 liter of LB medium. Cells were grown with vigorous shaking at 37 °C to an optical density at 600 nm (OD600) of 0.5 and were induced with 1 mM isopropyl-β-D-thiogalactopyranoside with 25 μM ZnSO4 for 6 h at 30 °C. Collected cells were resuspended with binding buffer (20 mM Tris–HCl (pH 7.9), 0.5 M NaCl, 1 mM TCEP {tris(2-carboxyethyl)-phosphine} and 5 mM imidazole) and cell extracts were prepared by sonication and ultra-centrifugation at 20,000 g for 30 min. Cell extracts were loaded onto a nickel-charged NTA column and then washed with six volumes of binding buffer followed by six volumes of washing buffer (20 mM Tris–HCl (pH 7.9), 0.5 M NaCl and 10 mM imidazole). Zur was eluted with 10 volumes of elution buffer (20 mM Tris–HCl (pH 7.9) and 0.5 M NaCl) containing linear imidazole gradients from 20 to 500 mM. Fractions containing Zur proteins were pooled and dialySed against buffer A (20 mM Tris–HCl (pH 7.8), 250 mM NaCl, 5% (vol/vol) glycerol and 4 mM EDTA)) to remove imidazole and nickel. For holo forms of Zur proteins, dialysed proteins in buffer A were further dialysed against buffer B (20 mM Tris–HCl (pH 7.8), 100 mM NaCl, 10% glycerol and 0.2 mM dithiothreitol (DTT)) and buffer C (20 mM Tris–HCl (pH 7.8), 50 mM NaCl, 30% glycerol, 1 mM DTT and 25 μM ZnSO4). The purified Zur proteins were concentrated by centrifugal filter devices (Millipore, 3,000 MW CO) before injection onto High load TM (16/60) pg Superdex G75 column in FPLC system (Pharmacia). The column was equilibrated with buffer G (20 mM Tris–HCl (pH 7.8), 50 mM NaCl, 2 mM DTT and 0.1 μM ZnSO4) so that the final buffer consisted of EMSA. Eluted fractions were monitored through ultraviolet detector. Concentrations of purified wild-type Zur and variant proteins were estimated in triplicates by Bradford assay (Bio-Rad) using bovine serum albumin (Sigma-Aldrich) as the calibration standard at A595. Measurement of ultraviolet absorbance at 280 nm, combined with calculated molar extinction coefficient (ɛ280=13,785 M−1 cm−1; http://expasy.org/cgi-bin/protparam), gave nearly identical values. The purity of each protein was confirmed through Coomassie blue staining of loaded protein samples in the SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel. The protein was stored in final storage buffer S (20 mM Tris–HCl (pH 7.8), 50 mM NaCl, 2 mM DTT, 30% glycerol and 0.1 μM ZnSO4) at −80 °C. To avoid metal contamination, all buffers were prepared through chelex-100 column (Bio-Rad). [...] Each Zur target promoter DNA probes of ∼45 bp containing Zur-binding sites were isolated using crush and soaking method from the polyacrylamide gel after annealing with each primer pairs in . The purified DNAs were labelled at 5′-ends with (γ-32P) ATP using T4 polynucleotide kinase. Binding reactions were performed with ∼1 fmol of labelled DNA fragments and 0.075–616 nM of purified Zur proteins in 20 μl of the reaction buffer (20 mM Tris–HCl (pH 6.4), 50 mM KCl, 1 mM DTT, 0.1 mg of bovine serum albumin per ml, 5% glycerol and 0.1 μg of poly(dI-dC), with 0.1 μM ZnSO4). Following incubation at room temperature for 20 min, the binding mixture was subjected to electrophoresis at 4 °C on a 5% polyacrylamide gel in TA (pH 6.4) buffer. After electrophoresis, the dried gels were exposed and quantified by a phosphor image analyser (Typhoon FLA 7000). A band intensity of unbound DNA probes was measured against Zur concentration using Multi Gauge V3.0 software. Digitalized data were fit to binding curves through SigmaPlot 2001 program (SPSS Inc.). Apparent Kd values, corresponding to the concentration of variables (Zur) at half-maximal upshift of DNA probes, were determined from at least three independent sets of experiments. […]

Pipeline specifications

Software tools MODELLER, PyMOL, ProtParam, SigmaPlot
Databases ExPASy
Applications Miscellaneous, Protein structure analysis, Protein physicochemical analysis
Organisms Bacteria, Bacillus subtilis
Chemicals Folic Acid, Zinc