油松谷胱苷肽转移酶Tau1单体结构稳定性研究

目的]探究油松PtGSTU1结构与功能的关系,探讨油松PtGSTU1蛋白的单体稳定性。方法]利用同源建模模拟PtGSTU1的三维结构,推测其N末端18位精氨酸(Arg18)和C末端103位天冬氨酸(Asp103)能够形成氢键来稳定蛋白单体结构。利用定点突变,分别将Arg18和Asp103突变为具有不同极性和构象的氨基酸残基,检测其蛋白的催化活性及结构稳定性。结果]6个Arg18突变体均无法获得高纯度的具有正确折叠的可溶蛋白,而Asp103突变体可以表达为可溶蛋白,Asp103突变体对不同底物的催化活性和亲和力明显低于野生型,对经典底物CDNB和GSH反应的催化速率(Vmax)降低了至少9倍,对底物的催化效率(kcat/Km)也明显降低。结论]Arg18和Asp103之间形成的氢键对稳定PtGSTU1单体结构具有重要作用,由于植物GST蛋白N端的保守性和C端结构域的多变性,Arg18的突变对结构和活性的影响大于Asp103,同时预示着C端结构域中可能存在其他氨基酸位点能够与18位精氨酸形成氢键,从而稳定蛋白单体折叠结构。

Monomer Structural Stability of a Tau Class Glutathione Transferase(PtGSTU1) from Pinus tabulaeformis

Objective] To study the relationship between structure stability and function of PtGSTU1. Method] Homology modeling was used to simulate the three-dimensional structure of PtGSTU1. It is proposed that arginine(Arg18) in N-terminal domain and aspartic acid 103(Asp103) in C-terminal domain are capable of forming a hydrogen bond to stabilize the protein monomer structure. Arg18 and Asp103 were mutated to amino acid residues with different polarities and conformations, respectively, using site-directed mutagenesis, and the catalytic activity and structural stability of the mutants were examined. Result] None of all the six Arg18 mutants could obtain high-purity soluble protein with the correct folding, while Asp103 mutant could be expressed as a soluble protein whose catalytic activity and structure stability to different substrates were significantly lower than those of the wild type. The catalytic rate( V max) of Asp103 mutant to classical substrates CDNB and GSH was reduced by 8/9 at least, and the catalytic efficiency( k cat/K m) to the substrate was also significantly reduced. Conclusion] The results proves that as the C-terminal domains are more variable than N-terminal domains in plant GST proteins, there may be some other amino acid sites in the C-terminal domains that can form hydrogen bonds with the arginine to stabilize the protein monomer.