【作者】 刘丹；

【导师】 王静云；

【作者基本信息】 大连理工大学 ， 生物化工， 2006， 硕士

【摘要】 蛋白结构的稳定及正确的折叠是它们执行各种生物功能的基础。蛋白质分子结构与功能的研究一直是生命科学中研究的热点。长期以来人们认为蛋白表面的氨基酸残基暴露在溶剂中，因而对蛋白结构的稳定性影响甚微。但最近几年研究成果对这一观念提出了质疑，发现蛋白表面带电氨基酸残基对蛋白的稳定性及性质具有非常大的影响，从而开辟了通过优化蛋白表面电荷与电荷的作用来调节蛋白结构稳定性及功能的新思路。 肌红蛋白是目前人类研究最多的蛋白质之一。曾有报道发现当将马心肌红蛋白表面44位氨基酸从天冬氨酸突变为赖氨酸后，突变肌红蛋白与细胞色素b5之间的电子转移速率提高了一个数量级，但这一蛋白表面氨基酸对马心肌红蛋白结构稳定性及功能的影响未见报道。 为了进一步了解肌红蛋白Mb表面44位天冬氨酸对肌红蛋白结构和功能的影响，本论文以野生肌红蛋白表达质粒pGYM为模板，设计特异突变引物，通过PCR定点突变的方法将肌红蛋白（Mb）基因上的第44位酸性氨基酸天冬氨酸（D）的密码子GAT变成碱性氨基酸赖氨酸（K）的密码子AAA，获得肌红蛋白突变体D44K的基因。将带有突变体的目的基因克隆在肌红蛋白表达质粒pGYM上，经热激转化大肠杆菌BL21，大肠杆菌发酵使突变蛋白D44K大量表达、收集菌体。酶法裂解细菌，依次用硫酸铵沉淀、离子交换柱层析、凝胶柱层析分离纯化突变蛋白，其纯度可达96％。用圆二色谱和紫外可见光谱分别研究野生型肌红蛋白及突变体（D44K）的耐热、耐酸及盐酸胍诱导的变性过程，实验结果表明：用碱性氨基酸Lys取代酸性氨基酸Asp44残基，增强了肌红蛋白耐热、耐酸变性能力，导致蛋白质的热变性中点温度Tm由71.9℃提高到75.1℃、酸变性中点（pH）_1／2从3.95降到3.88，但在盐酸胍溶液中，突变体易变性，其变性中点浓度Cm从1.88mM降为1.75mM。以愈创木酚为底物分别测定了不同条件下Mb（WT）和Mb（D44K）的过氧化物酶活性，实验结果表明：以愈创木酚为底物时，Mb（D44K）的Km为1.9mM而Mb（WT）的Km为3.3mM。Mb（WT）和Mb（D44K）过氧化物酶的最适温度均为50℃，在相同温度下，Mb（D44K）的过氧化物酶活性高于Mb（WT），且其过氧化物酶的热稳定性高于Mb（WT）。同时实验结果表明：Mb（D44K）过氧化物酶的最适pH为5.0，且在宽的pH范围内仍保持高过氧化物酶活性，而Mb（WT）过氧化物酶的最适pH5.5，在相同pH下，Mb（D44K）过氧化物酶对酸碱的稳定性高于Mb（WT），且两者在碱性溶液中其酶活性受到抑制。更多还原

【Abstract】 Structural stability and the right folding of proteins are important for their biological functions,and the relationships between structure and function for the proteins have been widely studied by many experts.Among different interactions that contribute to the protein stability（hydrophobic effect,hydrogen bonding,packing interactions,and hydration）,the effects of interactions between ionizable groups in the protein surface （charge-charge interactions） attracted little attention until recently.However, in past years, there have been a number of reports showing that charge-charge interactions on the protein surface are capable of modulating protein stability.Myoglobin is one of the most thoroughly studied proteins with respect to structure and function.It has been reported that when the Asp（D）44 on the surface of horse heart myoglobin is mutated to Lys（K）,the electron transfer rate between Mb（D44K） and Cytochrome b₅ was increase by ten fold.But there is no report about the influence of this residue on its stability and functions.To probe the effects of surface-charged residue Asp44 on the structure stability and functions of horse heart myoglobin,the code of Asp44,GAT in the gene of horse heart myoglobin was changed into AAA for Lys by PCR site-directed mutagenesis.The mutant gene was ligated into PstI/BamHI-cut pGYM and the resulting plasmid was transformed into E.coli BL21.The mutant protein was expressed in BL21 successfully.The bacteria containing mutant myoglobin were treated with lysozym.Then the mutant protein was purified by ammonium sulfate precipitation,ion-exchange chromatography and gel filtration,the recombinant Mb（D44K） was purified with 96% purity.Circular dichroism and UV-vis spectras were employed to monitor the defolding process of wild-type and mutant myoglobins under different temperatures,pH and the concentrations of guanidine hydrochloride.The Tm of Mb（D44K） is increased from 71.9℃ to 75.1℃ and （pH）_1/2 of Mb（D44K） is decreased from 3.95 to 3.88.The results show that the mutation of the surface-charged residue Asp44 to Lys44 increases the protein’s stability on its resistance to heat and pH.But mid-concentration of guanidine hydrochloride of Mb（D44K） is decreased from 1.88mM to 1.75mM for the protein denature.The Km of D44K for 2-methoxyphenol is 1.9 mM less than 3.3mM of wild type.The optimum temperature for the peroxidase activity of Mb（WT） and Mb（D44K） is 50℃. And Its optimum pH for the peroxidase activity of Mb（D44K） is decreased from 5.5 to 5.0. And the peroxidase activity of Mb（D44K） is higher than that of Mb（WT） at the same更多还原