对从头设计蛋白可折叠性的高效实验评估和改进

发布时间：2018-02-14 02:12

本文关键词： 蛋白质折叠性质蛋白质设计定向进化核磁结构解析　出处：《中国科学技术大学》2016年博士论文　论文类型：学位论文

【摘要】：蛋白质设计目前面临的最为棘手的一个问题就是怎样高效地挑选出折叠性质好的蛋白序列。对大量的设计蛋白序列都进行彻底的结构研究,既不现实也不值得。在这里,我们建立了一个可以有效地评估并且改进设计蛋白折叠性质的实验方法。该方法中,目的蛋白的结构稳定性与细菌的抗生素抗性呈正相关,在目的蛋白两端连上连接序列后,插入到TEM1-β-内酰胺酶中间,形成一个三明治样结构。折叠性质差的目的蛋白在周质腔中会被蛋白酶特异性地识别为非折叠蛋白进而被水解,表现为宿主细胞的低抗生素抗性。利用该系统不仅可以帮助我们评估设计蛋白的折叠性质,还可以在定向进化后筛选出能够弥补设计缺陷的突变体。我们以硫氧还蛋白的主链结构(PDB ID为1r26)作为设计模板,该蛋白具有113个氨基酸,形成了四个α-螺旋围绕着五个p-折叠的三维结构。在初次设计时,设计蛋白Dv_1r26在T EM1-β-内酰胺酶胞内系统显示出了很低的抗生素抗性,暗示着该设计蛋白的折叠性质差,与核磁一维氢谱实验结果一致。基于TEM1-β-内酰胺酶胞内系统对Dv 1r26进行了三轮定向进化,我们获得了一系列表现为高抗性的突变体,其中V31D,V61D和W105R这三个点突变在这些突变体中被频繁地观测到,我们选择突变体Dv_1r26_M1做为进一步的实验对象,该突变体仅含有四个点突变,包含被频繁观测到的三个位点突变和L23P。Dv_1r26_M1的一维氢谱和HSQC谱都具有很好的分散度,暗示着它的折叠性质良好。对Dv_1r26_M1进一步进行彻底的核磁结构实验分析可以看到：该蛋白的p-折叠片折叠性质良好,而且折叠片间的走向与设计结构一致,但是N端的α螺旋没有形成,C端的螺旋部分部分折叠。基于对初始设计序列可能存在的问题的分析,我们对设计方法进行了一些微小但十分重要的调整,使用修正后的设计模型设计出了新的蛋白序列,命名为E_1r26,它无需经过定向进化就在TEM1-β-内酰胺酶胞内系统中显示出了很高的抗生素抗性,随即对其进行了核磁结构解析。结果显示：计算出的E_1r26NMR模型与设计模板1r26高度一致,其中能量最低的模型与设计模板的主链重原子RMSD仅为1.7埃。此外,我们还测试了荧光蛋白Insertion Reporter系统对于蛋白质折叠性质的灵敏性。在对三个不同折叠性质的蛋白进行评估时,并没有看到明显的区别,无法做到对蛋白折叠性的有效评估。
[Abstract]:One of the most difficult problems in protein design is how to efficiently select protein sequences with good folding properties. It is neither realistic nor worthwhile to study thoroughly the structure of a large number of design protein sequences. We have established an experimental method that can effectively evaluate and improve the folding properties of the protein. In this method, the structural stability of the target protein is positively correlated with the antibiotic resistance of the bacteria. Inserted into the middle of TEM1- 尾 -lactamases to form a sandwich like structure. Proteins with poor folding properties are specifically recognized by protease as non-folded proteins in the periplasmic cavity and then hydrolyzed. Low antibiotic resistance in host cells. Using this system can not only help us assess the folding properties of design proteins, We used thioredoxin's main chain structure (PDB ID = 1r26) as the design template, and the protein had 113 amino acids. In the initial design, the design protein Dv_1r26 showed low antibiotic resistance in the intracellular system of T EM1- 尾 -lactamases, suggesting that the design protein had poor folding properties. In accordance with the results of nuclear magnetic one-dimensional hydrogen spectroscopy, we obtained a series of highly resistant mutants based on TEM1- 尾 -lactamase intracellular system for three rounds of directed evolution of Dv1r26. The three point mutations of V31D V61D and W105R were observed frequently in these mutants. We chose the mutant Dv_1r26_M1 as the further experiment object, the mutant contains only four point mutations. The one-dimensional hydrogen spectrum and HSQC spectrum of L23P.Dv26M1 and L23P.Dv26M1 have good dispersion. A further thorough analysis of the nuclear magnetic field (NMR) structure of Dv_1r26_M1 shows that the pfolding-sheet of the protein has good folding properties, and the orientation of the folding sheet is consistent with the design structure. But the a-helix of the N-terminal does not form the spiral part of the C-terminal. Based on the analysis of the possible problems in the initial design sequence, we have made some minor but very important adjustments to the design method. Using the modified design model, a new protein sequence was designed, named E _ S _ 1r _ (26), which showed high antibiotic resistance in the intracellular system of TEM1- 尾 -lactamases without directional evolution. The results show that the calculated 1r26 NMR model is highly consistent with the design template 1r26, and the RMSD of the main chain weight atom of the lowest energy model and the design template is only 1. 7 A. We also tested the sensitivity of the fluorescent protein Insertion Reporter system to the folding properties of proteins. When we evaluated the three proteins with different folding properties, we did not see obvious differences and could not effectively evaluate the folding properties of proteins.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q51

【相似文献】