Computational protocol: Purification, biochemical characterization and structural modelling of alkali-stable β-1,4-xylan xylanohydrolase from Aspergillus fumigatus R1 isolated from soil

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[…] The purified xylanase band from native-PAGE was cut and sent to National Centre for Biological Sciences (NCBS), Bangalore, India for mass-spectrometric analysis. The purified protein was subjected to in-gel digestion with additional reduction and alkylation to bring in better sequence coverage according to Shevchenko et al. []. Digested peptides were reconstituted in 15 μL of the 0.1 % formic acid and 1 μL was injected onto agilent 1200 nano flow HPLC system in-line coupled though advion nanomate to orbitrap discovery. Peptides were separated on Agilent ZORBAX SB-C18 nano column by a gradient developed from 1 % [v/v] acetonitrile, 0.1 % [v/v] formic acid to 80 % [v/v] acetonitrile, 0.1 % [v/v] formic acid in water over 70 min at a flow rate of 300 nl/min. Full MS in a mass range between m/z 300–2,000 was performed in the Orbitrap mass analyzer with a resolution of 30,000 at m/z 400 and an AGC target of 5 × 105. The strongest five signals were selected for CID (collision induced dissociation)-MS/MS in the LTQ ion trap at a normalized collision energy of 35 % using an AGC target of 3 × 104 and two microscans. Dynamic exclusion was enabled with one count during 30 s and an exclusion period of 180 s. The exclusion mass width was set to 0.01. For protein/peptide identification MS/MS data was searched against both uniprot swissprot amino acid sequence database (non-redundant database with reviewed proteins) and Uniprot TrEMBL database (database with unreviewed proteins) downloaded in August 2013 using an in-house mascot server (version 2.4) through Proteome Discoverer 1.3 software. The search was set up for full tryptic peptides with a maximum of three missed cleavage sites. Carbamidomethyl on cysteine, and oxidized methionine were included as variable modifications. The precursor mass tolerance threshold was 10 ppm and the maximum fragment mass error was 0.8 Da. The significance threshold of the ion score was calculated based on a false discovery rate of < 1 % estimated by the peptide validator node of the Proteome Discoverer software. Minimum of two high confident peptides were used as a prerequisite to identify the proteins. 12.5fmoles of Standard BSA digest was analyzed at the beginning and end of sequence to check the performance of the instrument. [...] Two programs were employed on the I-TASSER server [] to predict the secondary structure of the target protein xylanase: position specific iterated prediction (PSI-PRED) and position specific iterated-BLAST (PSI-BLAST). Initially, PSI-BLAST was performed for alignment of the query sequence against a non-redundant sequence database and then the secondary structure of the protein was predicted using PSI-PRED on the basis of the sequences generated by multiple alignments of the sequence homologs. LOMETS [] is a locally installed meta-threading program on I-TASSER server which combines ten state-of-art treading programs (MUSTER, FFAS-3D, SPARKS-X, HHSEARCH2, HHSEARCH1, Neff-PPAS, HHSEARCH, pGenTHREADER, wdPPAS and cdPPAS). The secondary structure of the query sequence predicted was then threaded through a library of PDB structures using LOMETS. The templates were ranked on the basis of sequence-based profile-profile alignment, structure-based profile- profile alignment, secondary structure match, backbone torsion angle match, solvent accessibility match and general hydrobhobicity scoring matrix. Top-ranked template hits were selected from each threading program for further analysis. [...] Two programs were used Support Vector Machine SVMSEQ [] and SPICKER [] for assembly and refinement of the target protein structure. SPICKER is clustering program which performs clustering using representative set of decoy confirmations. Unified knowledge-based force field has three components which guide simulations of structural assembly: (1) PDB Cα/ side chain correlations [], H-bonds and hydrobhobicity []; (2) spatial restraints of threading templates []; and (3) SVMSEQ sequence based contact predictions. There are two sets of simulations for determination of structural assembly. (1) Generation of initial structures for threading templates and (2) SPICKER generates cluster centroids from trajectories obtained in the first set of simulations. Finally, TM-align identifies the PDB structures which are structurally close to the cluster centroids []. [...] The program REMO [] depends on construction of full atomic models by optimizing H-bond network from C-alpha traces. Secondary structure specific backbone isomer was constructed using the program PSI-PRED. Full atomic model of our protein (xylanase) was constructed with reduced modeling simulation. Cα and side-chain centers play as the main simulation points for construction of models in reducing representation. These simulation points must be around to obtain better global topology of full atomic model with regular bond angle and bond length. In current study, Cα-RMSD and TM-scores parameters were considered for the verification of estimation of movement of Cα atoms relative to the native structure. Cα, C, N and O basic protein backbone fragments were matched from a secondary structure-specific backbone isomer library of non-redundant isomers from high-resolution PDB structures. H-bonding, clash/break-amendment, CHARMM22 [] and I-TASSER were the driving parameters in REMO refinement protocol. CHARMM22 identifies the potential energy functions by mathematical equations and respective force field dimensions of biomolecules. The models constructed by I-TASSER were ranked on the basis of structural density in SPICKER clusters. Eventually, the 3D models which had the highest scores were compared with the proteins of known structure and function in the PDB. Structural analogs of query protein in enzyme commission (EC) numbers, substrate binding site libraries and active site residues were evaluated on the basis of global and local structural similarity. […]

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