Computational protocol: Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92

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Protocol publication

[…] Proteins were produced in batch cultivations in E. coli BL21(DE3) in LB-medium with 25 mg/L kanamycin as antibiotic. Gene expression in the cells was induced with IPTG in the mid-log phase. The cells were recovered by centrifugation at 5000 × g for 10 min and washed once in binding buffer (sodium phosphate buffer pH 7.4) and then re-suspended in the same buffer. Cells were lysed by ultrasonication on ice 3 × 3 min in 3 min intervals and then centrifuged at 26700 × g for 20 min. Supernatants (containing His-tagged proteins) were purified by immobilized metal ion affinity chromatography 0MAC) (AKTA Prime, Uppsala, Sweden). Imidazole was used to elute bound protein. After elution, the proteins were dialyzed (3.5 kDa cutoff, SpectrumLab, CA) in 20 mM sodium phosphate buffer, pH 7.4, and 0.5 M NaCl at 4°C overnight and the following day by changing the dialysis buffer once. Purity of the protein solutions was determined by SDS–PAGE (Bio-Rad, Sweden) with 12.5% acrylamide. Protein concentration after purification was estimated spectrophotometrically at 280 nm. Theoretically calculated extinction coefficients were obtained using ProtParam ( and were 149,660 M/cm (full-length enzyme) and 82,850 M/cm (catalytic domain). [...] The three-dimensional structural models of the full-length protein were obtained by homology modeling, using the YASARA software ( The accuracy of the models obtained is supported by: (i) the relatively high identity in amino acid sequences, (ii) the molecular mechanics calculations including explicit solvent with molecules of water and (iii) the use of several crystallographic templates for generating the hybrid models, which in this case have higher Z-scores than using only one template.The target sequence contained 583 amino acids, of which the first 16 residues were excluded (including the 6× His tag used for purification). Two models were predicted, the first one as a single protomer and the second one as a homo-tetramer. The chosen modeling parameters are listed in Supplementary data, Table SI. The program built the model according to the alignment of the target sequence with several PDB templates from crystallographic structures. The templates were the crystallographic structures of β-xylosidases from three bacterial species: S ruminantium, G. stearothermophilus and B. halodurans (Table ). The 3D hybrid models obtained showed the lowest root mean square deviation (RMSD) to the G. stearothermophilus crystallographic structure (PDB 2EXI), despite lower overall sequence similarity (Table ). The overall RMSD of the atomic coordinates of the alpha carbons (Cα) was 0.896 Å when the hybrid model was superimposed with 2EXI. Details of the contribution of each template to the hybrid model are summarized in Supplementary data, Tables SII and SIII. The modeled loops were optimized and the side-chains fine-tuned by combining steepest descent and simulated annealing strategies for minimization. Half of the refined models were evaluated by quality Z-scores, which indicate how many standard deviations the model quality is away from the average high-resolution X-ray structure. Subsequently, a full unrestrained simulated annealing minimization was run for each model. Finally, to increase the accuracy, the best parts of the models generated were combined to obtain a hybrid model.The atomic coordinates of xylobiose (X2) were transferred from the crystallographic complex xylobiose-XynB3 (PDB: 2EXJ) from G. stearothermophilus () to the corresponding binding site in the model of WXyn43. After simulated annealing calculations of the resulting complex in explicit solvent with molecules of water, the interactions between the ligand and the protein model were compared with those of the crystal structure. […]

Pipeline specifications

Software tools ProtParam, YASARA
Databases ExPASy
Applications Drug design, Protein physicochemical analysis
Organisms Weissella cibaria, Escherichia coli
Chemicals Hydrogen