果蝇烯醇酶的结构生物学研究

发布时间：2018-01-14 08:28

本文关键词：果蝇烯醇酶的结构生物学研究　出处：《青岛科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：烯醇酶在细胞的许多生理生化功能方面都发挥着重要的调节作用,烯醇酶的差异化表达还被认为是一些病理状态的标志,诸如癌症、阿尔兹海默症、类风湿性关节炎等。本文以果蝇总cDNA为模板,克隆了烯醇酶基因,序列分析表明获得的基因与GenBank报道的果蝇烯醇酶序列完全一致。将含有该基因的表达载体转化大肠杆菌BL21感受态后,经SDS-PAGE检测证实,目的基因在BL21中得到了表达,获得了大小为47KD的蛋白,并具有较高的表达量,符合预期结果。在亲和层析纯化的基础上,进一步经过脱盐处理和凝胶过滤层析后获得纯度非常高的电泳纯目的蛋白,符合下一步的结晶要求。用座滴气相扩散法对果蝇烯醇酶蛋白进行大规模晶体筛选,初筛条件为10 mM CoCl2,0.1 M MES pH6.5,1.9 M(NH4)2SO4。晶体形成后再进一步优化,最终的优化条件为10 mM CoCl2,0.1 M MES pH6.5,1.9 M(NH4)2SO4,0.1 M CdCl2。优化后得到颗粒较大,生长良好的单晶。使用X射线晶体衍射的方法对优化后的晶体进行初步解析,在上海同步辐射光源的BLl7U光束工作站收集晶体衍射数据,用HKL2000进行数据处理。其分辨率可达2.1?,属于C121空间群,晶胞参数a=168.746?,b=119.210,c=103.191?,α=γ=90.00°,?=114.28°。果蝇烯醇酶的蛋白晶体结构由分子置换的方法确定。其结构解析表明,和其他大多数烯醇酶一样,果蝇烯醇酶能够在一个二聚体界面形成同型二聚体。果蝇烯醇酶的蝶形二聚体具有保守残基,并且通过离子键、氢键和疏水相互作用维持。它有一个保守的催化区域及一个带有不稳定L1环的相对活跃的构象。其活性位点被一个镉离子,两个钴离子和一个硫酸根离子占据,硫酸盐取代烯醇酶底物磷酸基团并且和残基Ala106,Arg440以及Ser441形成盐桥和氢键。与其他物种烯醇酶不同的是,果蝇烯醇酶在N末端有一超长区域,这一超长区域可能对其功能的发挥起着调节作用。本文首次对果蝇的烯醇酶(69-500)进行了结构解析,为进一步研究烯醇酶的结构和功能方面奠定了基础。
[Abstract]:Enolase plays an important role in regulating many physiological and biochemical functions of cells. The differential expression of enolase is also considered to be a marker of pathological state, such as cancer, Alzheimer's disease. In this paper, the enolase gene was cloned from the total cDNA of Drosophila melanogaster. Sequence analysis showed that the obtained gene was completely consistent with the sequence of Drosophila melanogaster enolase reported by GenBank. The expression vector containing the gene was transformed into E. coli BL21 competent state. It was confirmed by SDS-PAGE that the target gene was expressed in BL21, and a protein of 47KD was obtained. In accordance with the expected results, on the basis of affinity chromatography purification, after further desalination and gel filtration chromatography, a very high purity electrophoresis pure protein was obtained. According to the requirements of the next step of crystallization, the large scale crystal screening of drosophila enolase protein was carried out by gas phase diffusion method. The initial screening condition was 10 mm CoCl2 + 0.1 M MES pH6.5. The crystal was further optimized after the crystal formation, and the final optimization condition was 10 mm CoCl2 + 0.1 M MES pH6.5. 1.9 NH _ 4H _ 4SO _ 4SO _ 4O _ (1) M CdCl _ (2). The particle size was larger after optimization. The X-ray diffraction method was used to analyze the optimized crystal and the diffraction data were collected at the BLl7U beam workstation of Shanghai Synchrotron radiation source. Data processing with HKL2000. Its resolution can reach 2.1? , belonging to C121 space group, unit cell parameter a168.746? BX 119.210CU 103.191? , 伪 = 90.00 掳? The protein crystal structure of Drosophila enolase was determined by the method of molecular replacement. The structural analysis showed that the protein crystal structure was the same as most other enolases. Drosophila enolase can form a homodimer at the interface of a dimer. The butterfly dimer of Drosophila enolase has conserved residues and passes through ionic bonds. The hydrogen bond and hydrophobic interaction are maintained. It has a conserved catalytic region and a relatively active conformation with unstable L1 ring. Its active site is a cadmium ion. Two cobalt ions and one sulfate ion occupy the sulfate-substituted enolase substrate phosphate group and the residue of Ala106. Arg440 and Ser441 form salt bridges and hydrogen bonds. Unlike other species enolase, Drosophila enolase has a very long region at the N end. This super-long region may play a role in regulating its function. In this paper, the structure of the enolase of Drosophila melanogaster 69-500 was analyzed for the first time. It lays a foundation for further study on the structure and function of enolase.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55

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