Effect of ion pair on thermostability of F1 protease: integration of computational and experimental approaches.
A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.
Enzyme and Microbial Technology Research Group, Selangor, Malaysia. rnzaliha@biotech.upm.edu.my
Raja Noor Zaliha Raja Abd
RN
Rahman
Noor Dina
ND
Muhd Noor
Noor Azlina
NA
Ibrahim
Abu Bakar
AB
Salleh
Mahiran
M
Basri
D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D007477:Ions / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D057057:Serine Proteases / Q000737:chemistry* / Q000378:metabolism
D007477:Ions; D050505:Mutant Proteins; D057057:Serine Proteases