6pak | pdb_00006pak

Insight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis

spinqualitylabelsall models 6pak, resolution 1.98Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6pak is a 2 chain structure with sequence from Bacillus subtilis subsp. subtilis str. 168. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.98Å Ligands: CA, EDO Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function SUBT_BACSU Subtilisin is an extracellular alkaline serine protease, it catalyzes the hydrolysis of proteins and peptide amides. Publication Abstract from PubMed Rational design-guided improvement of protein thermostability typically requires identification of residues or regions contributing to instability and introduction of mutations into these residues or regions. One popular method, B-FIT, utilizes B-factors to identify unstable residues or regions and combines them with other strategies, such as directed evolution. Here, we performed structure-based engineering to improve the thermostability of the subtilisin E-S7 (SES7) peptidase. The B-value of each residue was redefined in a normalized B-factor calculation, which was implemented with a refined bioinformatics analysis strategy to identify the critical area (loop 158-162) related to flexibility and to screen for suitable thermostable motif sequences in the PDB database that can act as transplant loops. In total, we analyzed 445 structures and identified 29 thermostable motifs as candidates. Using these motifs as a starting point, we performed iterative homologous modeling to obtain a desirable chimera loop and introduced five different mutations into this loop to construct thermostable SES7 proteins. Differential scanning fluorimetry (DSF) revealed increases of 7.3 degrees C in the melting temperature of an SES7 variant designated M5 compared with the wild-type. The X-ray crystallographic structure of this variant was resolved at 1.96 A resolution. The crystal structure disclosed that M5 forms more hydrogen bonds than the wild-type protein, consistent with design and molecular dynamics simulations (MDS) results. In summary, the modified B-FIT strategy reported here has yielded a subtilisin variant with improved thermostability and promising industrial applications, supporting the notion that this modified method is a powerful tool for protein engineering. Enhancing subtilisin thermostability through a modified normalized B-factor analysis and loop-grafting strategy.,Tang H, Shi K, Shi C, Aihara H, Zhang J, Du G J Biol Chem. 2019 Oct 15. pii: RA119.010658. doi: 10.1074/jbc.RA119.010658. PMID:31615894 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Subtilisin 3D structures

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