發(fā)布時(shí)間：2019-05-28 18:23

【摘要】：藍(lán)銅蛋白(Rusticyanin)是第I類(lèi)銅蛋白家族成員，是氧化亞鐵硫桿菌中鐵呼吸電子鏈中重要的組分。該蛋白分子內(nèi)的銅原子與Cys138，His85、His143以強(qiáng)健結(jié)合，與Met148以較弱的鍵結(jié)合，，形成四面體結(jié)構(gòu)。Cys138和His85只是維持銅與蛋白結(jié)合的關(guān)鍵位點(diǎn)，Met148位點(diǎn)軸向配體對(duì)于氧化還原電子傳遞有一定的影響，但并不是控制氧化還原反應(yīng)的關(guān)鍵位點(diǎn)，His143位點(diǎn)目前對(duì)其的研究還很少，很可能是氧化還原的開(kāi)關(guān)，但目前沒(méi)有相關(guān)文獻(xiàn)報(bào)道過(guò)。 本論文從嗜酸亞鐵氧化硫桿菌中克隆出藍(lán)銅蛋白基因，構(gòu)建了藍(lán)銅蛋白重組質(zhì)粒，并將其在大腸桿菌載體中表達(dá)，用親和一步層析方法純化。光譜學(xué)數(shù)據(jù)表明其能夠和IRO蛋白發(fā)生反應(yīng)，證明了目前亞鐵氧化系統(tǒng)電子傳遞的途徑：IRO和藍(lán)銅蛋白之間發(fā)生了電子傳遞。 本論文構(gòu)建了His143位點(diǎn)的定點(diǎn)突變質(zhì)粒，并將其在大腸桿菌中表達(dá)用鎳柱純化。通過(guò)聚丙烯酰胺凝膠電泳，光譜學(xué)數(shù)據(jù)確定143位點(diǎn)是藍(lán)銅蛋白與外界電子傳遞的關(guān)鍵位點(diǎn)，移除其結(jié)構(gòu)上與銅連接的咪唑基團(tuán)導(dǎo)致結(jié)合的銅減少，若繼續(xù)在咪唑溶液中孵育則藍(lán)銅突變蛋白活性有所增加。分子結(jié)構(gòu)模型證明若將咪唑基團(tuán)突變后，銅離子就暴露下溶液環(huán)境中，外面的溶劑很容易進(jìn)去到四面棱形結(jié)構(gòu)里導(dǎo)致銅離子結(jié)構(gòu)瓦解，電子不發(fā)生傳遞。因此His143位點(diǎn)是藍(lán)銅蛋白電子傳遞的關(guān)鍵部位。
[Abstract]:Blue copper protein (Rusticyanin) is a member of class I copper protein family and an important component of iron respiratory electron chain in thiobacillus ferrooxidans. The copper atoms in the protein molecule bind strongly to Cys138,His85,His143 and weak bond to Met148 to form tetrahedral structure. Cys138 and His85 are only the key sites to maintain copper binding to protein. The axial ligand of Met148 site has a certain effect on redox electron transfer, but it is not the key site to control the redox reaction. At present, there is little research on it at His143 site, which may be the switch of redox. However, no relevant literature has been reported at present. In this paper, the blue copper protein gene was cloned from thiobacillus ferrooxidans, and the recombinant plasmid of blue copper protein was constructed and expressed in E. coli vector. The recombinant plasmid was purified by affinity one step chromatography. Spectroscopic data show that it can react with IRO protein, which proves that electron transfer takes place between IRO and blue copper protein in ferrous oxidation system at present. In this paper, the site-directed mutant plasmid of His143 site was constructed and purified by nickel column. Through polyacrylamide gel electrophoresis, the 143 site was determined to be the key site for the electron transfer between blue copper protein and the outside world, and the removal of imidazole groups linked to copper resulted in the decrease of copper binding. If incubated in imidazole solution, the activity of blue copper mutant protein increased. The molecular structure model shows that if the imidazole group is mutated, the copper ion will be exposed to the solution environment, and the external solvent can easily go into the tetrahedral structure and lead to the collapse of the copper ion structure, and the electron does not transfer. Therefore, His143 site is the key site of electron transmission of blue copper protein.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R341

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