Structure and Activity of Sulfide:Quinone Oxidoreductase and Its Mutants from Thiobacillus denitrificans ATCC25259
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Abstract:
The wild-type protein and mutant protein models of sulfide:quinone oxidoreductase (SQR) from Thiobacillus denitrificans ATCC25259 were generated by homology modeling using Discovery Studio 3.5. All models were optimized by molecular mechanics and molecular dynamics using GROMACS 5.1.2, yielding protein models in a stable state with a relatively low energy. Three methods, i.e., PROCHECK, Verify 3D, and ProSA, were used to evaluate the models, and the results indicated that the models were reasonable and reliable. The protein models were used to calculate the interactions between proteins and ligands, as well as the solvent accessible surface (SAS) and energy values. The expression vectors of SQR and mutants were constructed and transferred into Escherichia coli BL21 (DE3) to express the proteins with a molecular weight of about 65 ku. Several wild-type and mutant proteins containing the 6× His tag were purified by nickel-affinity chromatography. The SQR enzyme activity was determined using established methods, and the results showed that the activity of the mutants was lower than that of the wild-type SQR. Both simulation calculations and experimental verification suggested that the structural stability of the alpha helix in the C-terminal of SQR had an important influence on the structural stability of the protein. If the structural stability of the protein decreased, the enzyme activity also decreased.
