斑馬魚粒線體型絲胺酸羥基甲基轉移酵素之表現、純化以及結構特性分析

Expression, Purification and Structural Characterization of Zebrafish Mitochondrial Serine Hydroxymethyltransferase

陳奕宏(Yi-Hung Chen)

指導教授 ： 陳俊榮

國立清華大學/理學院/物理學系/碩士(2013年)

摘要

絲胺酸羥基甲基轉移酵素(SHMT)普遍存在自然界中，包含原核生物和真核生物體內，催化許多重要的新陳代謝，包含葉酸代謝。許多生物的絲胺酸羥基甲基轉移酵素已經被廣泛研究其功能，有研究指出該酵素與相關代謝失衡將導致許多疾病，包含癌症，因此絲胺酸羥基甲基轉移酵素具有發展相關藥物的潛力。斑馬魚是近年來興起的實驗動物模型，其絲胺酸羥基甲基轉移酵素已經藉由大腸桿菌大量表現純化，並研究其生化功能，然而尚未有任何結構資訊。在這篇論文中，我們大量表現與純化了斑馬魚的粒線體型絲胺酸羥基甲基轉移酵素(zmSHMT)，藉由動態光散射粒徑分析儀(DLS)來偵測蛋白質溶液的同質性，同步輻射圓二色圖譜儀(SRCD)來偵測蛋白質中二級結構的比例，以及小角度 X 光散射實驗(SAXS)得到蛋白質在溶液中的低解析度模型與動態結構資訊。結合以上實驗結果，我們發現比起其他已知其蛋白質結構的真核生物(包含人類、兔子、老鼠)，斑馬魚的粒線體型絲胺酸羥基甲基轉移酵素具有較多 $eta$-摺板的二級結構，以及較大的巨分子粒子半徑，同時發現由斑馬魚的粒線體型絲胺酸羥基甲基轉移酵素，四個單聚體結合成四聚體的三級結構，具有較為拉長的結構形狀，因而與其他哺乳類酵素在結構特性上有所不同。

關鍵字

斑馬魚 ； 絲胺酸羥基甲基轉移酵素 ； 同步輻射圓二色圖譜儀 ； 動態光散射粒徑分析儀 ； 小角度 X 光散射實驗

並列摘要

Serine hydroxymethyltransferase (SHMT), widely distributed in nature including Prokaryotic and Eukaryotic organisms, catalyzes several important biosynthesis reactions, such as in folate metabolism. Functions of SHMTs have been extensively studied in many living systems, and enzymic imbalance in serine and folate metabolism have been reported to cause diseases including cancer. SHMT can be target for designing and developing inhibitors which could be used as anticancer drugs. The zebrafish has risen to be a prominent animal model because it is easy to grow and breed and has comparable organs and tissues with mammals. Zebrafish Mitochondrial Serine Hydroxymethyltransferase(zmSHMT) has been expressed in E. coli and studied, but structural information remains unknown. In this thesis, Dynamic Light Scattering(DLS) serves as homogeneity indicator during purification, Synchrotron Radiation Circular Dichroism(SRCD) helps identifying secondary structures, and Small-Angel X-ray Scattering(SAXS) helps calculating hydrodynamic radius information and resolving low resolution structural information in solution. With above results, zmSHMT shows more $eta$-sheet secondary structure and larger size than that of other existing structures of Eukaryotic species including human, rabbit and mouse, which also imply elongated tertiary structure that may lead to differences from mammals.

並列關鍵字

Zebrafish ； Serine Hydroxymethyltransferase ； SRCD ； DLS ； SAXS

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