Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/16629
標題: 探究新抑癌素蛋白結合穴口底部摺板與生色團之交互作用
Probing the Key Interactions on the Bottom-Strands of Binding Cleft in Neocarzinostatin
作者: 李英賢
李英賢, Ying-Hsien Lee
關鍵字: neocarzinostatin;新抑癌素
出版社: 化學系所
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摘要: 
新抑癌素(neocarzinostatin)是一種天然且強力的抗腫瘤抗生素,它包含一個全β摺板的蛋白(aponeocarzinostatin)和一個以非共價作用與此蛋白結合的生色團(chromophore)。新抑癌素生色團並不安定,蛋白扮演著攜帶者和保護者的角色,可避免生色團分解,還可調控操縱生色團的釋放、攜帶生色團進入腫瘤細胞等作用。釋放的生色團被硫醇激活後,得以進行芳香環化反應,形成相當不穩定的雙自由基中間物,該自由基中間物具有生物毒性,能抓取DNA中五碳醣上的氫原子,使腫瘤細胞DNA受損達到毒殺細胞的功效。
生色團與新抑癌素蛋白結合時,生色團上的萘環(naphthoate moiety)位於新抑癌素蛋白疏水性結合穴口的底部,是生色團與新抑癌素蛋白緊密結合不可或缺的部份,另外,此萘環和穴口底部的胺基酸殘基有許多特定的交互作用;這些交互作用是新抑癌素蛋白能與生色團結合並具穩定生色團功能的要素。
為探討新抑癌素蛋白穴口底部的胺基酸殘基對結合生色團的影響,我們利用定點突變技術製造突變質體DNA,轉殖入大腸桿菌後大量表現所需的突變蛋白;以下四個新抑癌素蛋白殘基V34,Q36與Q94,L97分別兩兩位於構成新抑癌素蛋白穴口底部的兩個不同β摺板中,其骨架或側鏈與生色團上萘環相距不遠,我們檢驗生色團釋放速率是否因此突變而改變,以便探討蛋白與生色團結合強度是否因突變而改變。由大腸桿菌大量表現且純化後的突變蛋白V34A、Q36A、Q94A和L97A,以質譜儀鑑定分子量無誤且雙硫鍵均完整存在,以圓二色光譜儀(Circular Dichroism)證明它們的二、三級結構與新抑癌素蛋白相似,我們並比較突變蛋白和新抑癌素蛋白的熱穩定性。之後用純化的重組突變蛋白與淬取的生色團再結合形成複合體,利用螢光光譜儀分析比較突變蛋白與新抑癌素蛋白釋放生色團速率的差異,本研究結果顯示突變蛋白V34A釋放生色團的速率大幅提高,推測這特定蛋白殘基大幅影響生色團結合的強度;而L97A蛋白釋放生色團速率與天然新抑癌素接近。
利用高效能液相層析分析殘餘生色團比例後,我們確認其突變蛋白釋放生色團趨勢合乎螢光動力學實驗;所得的實驗結果希望能提供些許線索,使我們了解新抑癌素蛋白結合穴口底部殘基影響生色團結合強度的機理。

Neocarzinostatin, a natural and potent antitumor antibiotic chromoprotein complex, is composed of an all β-sheet protein (aponeocarzinostatin) and a labile enediyne containing chromophore. Aponeocarzinostatin acts as a carrier and protector of the bioactive chromophore and plays crucial roles in regulating availability of the chromophore. Released chromophore can be activated by thiols in vivo and in vitro after which, diradical intermediate is formed via Bergman cycloaromatization. To perform its cytotoxic function, the radical intermediate abstracts hydrogen from deoxyribose backbone to cause DNA damages in the presence of oxygen.
Specific intermolecular interactions between the chromophore and aponeocarzinostatin, in particular those amino acid residues at the bottom of the binding pocket of aponeocarzinostatin, underlie the binding and stabilization of the labile chromophore. How the non-covalent interactions between protein and its chromophore lead to very tight binding (KD ≤ 0.1 nM) raises an interesting question. Therefore, understanding the interaction of the chromophore with individual residues in the protein binding pocket is crucial for such investigation.
In the present study, we employed alanine scanning mutagenesis on four amino acid residues (V34, Q36, Q94 and L97) located in different β-sheet strands at the bottom of the binding pocket of aponeocarzinostatin. To investigate their contribution to the overall strength of binding interactions, a PCR-based in vitro site-directed mutagenesis was adapted to produce mutant protein expression constructs. Mutant recombinant aponeocarzinostatin (V34A, Q36A, Q94A and L97A) proteins were expressed in E. coli followed by purification involving various chromatographic techniques. Purified mutant proteins were subjected to circular dichroism spectroscopic studies to analyze secondary and tertiary structures. Structural stability of the mutant proteins is investigated by thermal denaturation experiment monitored by circular dichroism. Reconstituted holoneocarzinostatin for each mutant was prepared by titrating each mutant protein with 1:1 molar ratio of the extracted chromophore. Influence of mutant proteins on the binding interactions was studied by examining release rate of the chromophore. Fluorescence analysis of chromophore release kinetics reveals that the release rate of V34A is significantly higher than that of wild-type neocarzinostatin. Mutant L97A had similar release rate as that of the wild-type neocarzinostatin. Chromophore release trend among the mutants was further confirmed by HPLC analysis.
The results offer some clues for understanding specific role of some residues at the bottom of binding cleft in intermolecular interactions between aponeocarzinostain and its chromophore.
URI: http://hdl.handle.net/11455/16629
其他識別: U0005-2512200710563501
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