本文研究了通过Discovery Studio 3.5利用同源建模法构建了脱氮硫杆菌ATCC25259的硫化物:醌氧化还原酶（Sulfide:quinone oxidoreductase，SQR）野生型蛋白及突变体蛋白模型，通过GROMACS 5.1.2对所有模型进行分子力学及分子动力学的优化，使蛋白模型处于能量较低且结构稳定的状态。使用PROCHECK，Verify 3D和ProSA三种模型评价方法对模型进行评价，表明蛋白模型具有较高的合理性。使用该蛋白模型计算蛋白相互作用、SAS及能量值。将构建好的SQR及突变体的表达载体转入大肠杆菌BL21（DE3）诱导表达分子量约65 ku的蛋白。使用镍柱亲和层析纯化经大量表达含有6×His标签的野生型与突变体蛋白，采用已经建立的SQR活性测定方法，进行酶活性测定实验，结果表明突变体酶活性较低。从模拟计算与实验验证两方面说明SQR C端α螺旋结构对蛋白的结构稳定性具有重要影响，蛋白结构稳定性降低，从而酶活性降低。

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.

广州市产学研协同创新重大专项（201508020110）