人类Smad7的N端结构域（NTD）蛋白质的折叠性质研究

发布时间：2018-01-05 02:02

本文关键词：人类Smad7的N端结构域（NTD）蛋白质的折叠性质研究　出处：《上海交通大学》2011年硕士论文　论文类型：学位论文

【摘要】：Smad7是一种重要的抑制型Smad蛋白,它主要通过竞争受体调控型Smad与细胞膜上I型TGF-β受体之间的结合,来调节TGF-β受体的泛素化和降解,对TGF-β信号通路起负调节作用。Smad7的N端结构域(NTD)在介导Smad7与E3泛素连接酶Smurf2和E2泛素结合酶UbcH7的相互作用过程中起关键作用,但是我们对它的生物化学和结构生物学特性知之甚少。本论文首先采用生物信息学方法对人类Smad7氨基酸序列的折叠倾向性进行分析,并且结合二级结构预测和多物种间抑制型Smad序列对比得到预测的Smad7-NTD蛋白的性质特征。然后我们构建一系列删除不同长度N端序列和中间预测Loop序列的重组蛋白,对这些蛋白进行生物化学特性分析。结果显示:Smad7的N端前20个左右的氨基酸影响Smad7-NTD蛋白的可溶性,它的删除会造成蛋白质不溶或聚集,因此推测这些氨基酸残基在蛋白质折叠中有非常重要的作用。同时由生物信息和生物化学实验的结果可以看出,Smad7-NTD的N端存在一个不参与折叠的无规Loop结构,这个Loop的存在导致Smad7-NTD蛋白易降解,而删除这段Loop可以提高蛋白质的稳定性,使蛋白更适用于后期结晶实验的研究。同时本论文对另一种抑制型Smad蛋白Smad6进行了生物信息学分析,发现其结构预测与Smad7相似,但是对其蛋白质的性质还有待进一步研究。同时也对序列与其他R-Smad高度同源,但是功能却不一样的Smad2-MH1结构域进行了纯化与初步结晶研究,表达了删除部分氨基酸残基且性质稳定、可用来做结晶的Smad2-MH1重组蛋白。
[Abstract]:Smad7 is an important inhibitory Smad protein, which binds to type I TGF- 尾 receptor mainly through competitive receptor regulated Smad. To regulate the ubiquification and degradation of TGF- 尾 receptors. The N-terminal domain NTD of Smad7 plays a negative role in TGF- 尾 signaling pathway. It plays a key role in mediating the interaction of Smad7 with E3 ubiquitin ligase Smurf2 and E2 ubiquitin binding enzyme UbcH7. But little is known about its biochemical and structural biological properties. Firstly, the folding tendency of human Smad7 amino acid sequence was analyzed by bioinformatics. Combining the prediction of secondary structure with the comparison of multiple species inhibitory Smad sequences, we obtained the properties of predicted Smad7-NTD proteins. Then we constructed a series of N-terminal sequences and intermediate sequences with different lengths. To predict the recombinant protein of Loop sequence. The results showed that the first 20 amino acids in the N-terminal of Smad7 affected the solubility of Smad7-NTD protein. These amino acid residues may play a very important role in protein folding and can be seen from the results of bioinformatics and biochemistry experiments. There is a random Loop structure in the N-terminal of Smad7-NTD which does not participate in folding. The existence of this Loop leads to the degradation of Smad7-NTD protein. The removal of this Loop can improve the stability of the protein and make the protein more suitable for later crystallization experiments. At the same time, the bioinformatics analysis of another inhibitory Smad protein Smad6 was carried out in this paper. It was found that the structure prediction of Smad6 was similar to that of Smad7. However, the properties of its protein still need to be further studied, and the sequence is highly homologous to other R-Smad. However, the Smad2-MH1 domain with different function was purified and crystallized, and some amino acid residues were deleted and the properties were stable. Smad2-MH1 recombinant protein can be used to make crystal.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341

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