Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/15813
DC FieldValueLanguage
dc.contributor.advisor金德航zh_TW
dc.contributor.advisorDer-Hang Chinen_US
dc.contributor.author羅羽華zh_TW
dc.contributor.authorLuo, Yu-Huaen_US
dc.date2005zh_TW
dc.date.accessioned2014-06-06T06:54:35Z-
dc.date.available2014-06-06T06:54:35Z-
dc.identifier.urihttp://hdl.handle.net/11455/15813-
dc.description.abstract新抑癌素 (Neocarzinostatin, NCS)為一種抗腫瘤抗生素,由鏈黴菌屬 Streptomyces carzinostaticus 所分泌製造並從中分離出來,它包含了一個具有生物活性的不穩定烯雙炔生色團 (Chromophore, NCS-Chr)和113個氨基酸所組成的β摺板 (β-sheet)蛋白 (ApoNCS)。新抑癌素蛋白對於生色團有保護和穩定的效果,並有調控操縱生色團的釋放、攜帶生色團進入腫瘤細胞及改變生色團環化路徑等用途。當生色團從蛋白中被釋放出來時,烯雙炔九元環會因為受到硫醇的激活而產生雙自由基,抓取DNA五碳糖上的氫原子使DNA受傷斷裂。由NCS複合體晶體結構顯示,生色團的主要活性部位 - 烯雙炔九元環 - 靠近蛋白的第78個氨基酸殘基,即苯丙胺酸 (Phe) 側鏈上的苯環,兩者所產生的π-π分子內交互作用力推測可以穩定生色團 (Kim et al. 1993)。由已發表的NMR研究顯示,Phe78側鏈在水溶液中有多種不同的構形 (Imajo et al. 1995),基於這些理由,加上我們實驗室目前已有的數據,已知Phe78對於生色團的釋放扮演一個重要的角色。目前我們研究目標是闡明Phe78支鏈對於生色團釋放的影響,我們用單點突變蛋白的技術,置換第78個氨基酸苯丙胺酸,以帶正電荷 (F78K)、負電荷 (F78E)、親水性 (F78Q)、氫原子 (F78G)和苯環 (F78Y)側鏈的氨基酸殘基取代原本的苯丙胺酸,希望藉由這些變異體 來解釋釋放機制。 我們利用PCR進行生物體外的突變作用,建構apoNCS第78個氨基酸殘基的變異體。突變蛋白基因序列經由DNA定序分析確認,重組蛋白經由IPTG誘發E. coli表現,並以蛋白質電泳 (SDS-PAGE)、高效能液相層析儀 (HPLC)、質譜儀 (LCQ mass)、及雙硫鍵形成測試 (Iodo-test)來鑑定突變蛋白及其純度。之後我們使重組蛋白和生色團再結合成複合體 (NCS, holoNCS)以進行螢光動力學實驗,測量蛋白釋放生色團初始速率,我們發現有些突變蛋白的釋放速率,如F78K,遠大於天然蛋白 (wild type, W.T.),雖然Tm熱穩定度實驗顯示重組過的突變蛋白熱穩定性和W.T.一樣好,圓二色旋光光譜儀 (circular dichroism, CD)和NMR 2D 15N-H1 HSQC鑑定這幾種製備的突變蛋白,發現所有的二級及三級結構也幾乎和天然蛋白相同,蛋白的活性穴口部位 (binding site)和整體骨架沒有因為突變第78個氨基酸位置而有所變動,所以我們認為影響生色團釋放的原因,應該和Phe78上的側鏈性質有關,未來會進一步以NMR技術檢測,也會針對各種推測做更多的突變蛋白來加以驗證,以闡明新抑癌素蛋白釋放活性生色團的機制。zh_TW
dc.description.abstractNeocarzinostatin, an antitumor antibiotic from Streptomyces carzinostaticus, is composed of a labile enediyne chromophore and an all-β protein with 113 amino acid residues. The biological activity in damaging DNA is a sole property of the chromophore. To perform its function, the chromophore needs to be released from the protein. The crystal structure of the chromoprotein complex shows that the pi-face of the enediyne ring in the chromophore interacts closely with the Phe78 benzene ring side chain in the protein. The reported NMR studies show that the side chain of Phe78 has multiple conformations in solution. Our accumulated data suggest that Phe78 may have an important role in controlling the release of the chromophore. The present investigation aims at elucidating how Phe78 is involved in the chromophore release. Here we use apoNCS variants at the 78th amino acid residue with alternative side chains to serve such a purpose. In this study, we choose 5 different variants with 78th side chain that is absent (G), positively charged (K), negatively charged (E), neutral but highly polared (Q), or similarly structured (Y). In the present study, a PCR based in vitro mutagenesis method is used to construct apoNCS variants at Phe78 position. The mutant apoNCS genes are sequenced to confirm the intended mutation at the desired position. The recombinant wild-type apoNCS and its variants are purified from IPTG-induced expression of respective proteins from in E. coli The identity and purity of apoNCS variants are analyzed by SDS-PAGE, HPLC, mass spectroscopy, and disulfide linkage examination. The fluorescence-based kinetic release experiments with reconstituted holo-proteins are performed for each purified mutants as well as the recombinant wild-type protein. Measurements of initial rate reveal that some mutants, in particular F78K, can have several fold faster rate to release its chromophore than WT can. On the other hand, the circular dichroism studies show that all these mutants have similar secondary as well as tertiary structure to that of WT protein. Tm measurements suggest that thermal stability of these mutants are similar. The NMR 2D 15N-H1 HSQC experiments further confirm the close structural similarity between mutants and WT. Our results suggest that the faster chromophore release rate could be attributed to varied chemical nature of the side chain being replaced at Phe78 position, but not from significant backbone structural changes. Further NMR studies may throw in more light on the mechanism of the chromophore release.en_US
dc.description.tableofcontents目 錄 CONTENTS 英文摘要 ENGLISH ABSTRACT 1 中文摘要 CHINESE ABSTRACT 3 第一章 緒論 INTRODUCTION 5 第二章 材料與方法 MATERIALS AND METHODS 17 第三章 實驗結果 RESULTS 50 第四章 結果討論 DISCUSSION 57 第五章 總結 CONCLUSION 60 參考文獻 REFERENCES 62 圖形 FIGURE 68 表格 TABLE 102 反應機制 SCHEME 104 附錄 APPENDIX 106 圖 目 錄 FIGURE Fig. 1 新抑癌素結構示意圖 68 Fig. 2 九元環烯雙炔結構的抗腫瘤抗生素 69 Fig. 3 十元環烯雙炔類的抗腫瘤抗生素 70 Fig. 4 新抑癌素生色團的活化作用 6 Fig. 5 激活後的生色團產生雙自由基攻擊DNA鏈 7 Fig. 6 NCS-Chr誘導DNA鏈裂解或受損機制 8 Fig. 7 新抑癌素蛋白結構顯示圖 9 Fig. 8 ApoNCS胺基酸序列 10 Fig. 9 NCS Chromophore結構 11 Fig. 10 Chromophore A、B & C 12 Fig. 11 產物一 & 產物二 13 Fig. 12 NCS三度空間結構圖 14 Fig. 13 Conformational difference of Phe78 15 Fig. 14 NCS蛋白第78位置所置換的氨基酸殘基 16 Fig. 15 Agarose gel (A) F78E (B) F78Q 71 Fig. 16 經核酸定序實驗室鑑定後所得正確DNA序列 72 Fig. 17 由SDS-PAGE鑑定重組蛋白 80 Fig. 18 Calmodulin affinity column層析圖 81 Fig. 19 由SDS-PAGE鑑定EK反應的時間 82 Fig. 20 DEAE ion-exchange column層析圖 83 Fig. 21 (A)重組蛋白的UV圖 (以F78Q為例) (B)文獻資料 84 Fig. 22-1 以HPLC分析天然NCS蛋白純度 85 Fig. 22-2 以HPLC分析重組突變蛋白純度 (以WT為例) 86 Fig. 23 Comparison of CD spectra of recombinant apoNCS 87 Fig. 24 比較重組蛋白在不同濃度下的CD測量值 88 Fig. 25 利用CD分析再結合過的holo-protein 89 Fig. 26 Mass spectra of apoNCS variants 90 Fig. 27 新抑癌素蛋白胺基酸序列 54 Fig. 28 經過Iodoacetamide reaction後所得到的質譜圖 93 Fig. 29 螢光動力學實驗設計原理 55 Fig. 30 螢光動力學實驗:HoloNCS濃度對時間作圖 95 Fig. 31 Ln [HoloNCS]對時間做圖 (以F78K為例) 96 Fig. 32 以CD鑑定重組蛋白的熱穩定溫度Tm 97 Fig. 33 以CD鑑定重組蛋白二級和三級結構的熱穩定溫度 98 Fig. 34 利用CD觀測新抑癌素重組蛋白二級結構的開散狀態 99 Fig. 35 2D-NMR HSQC光譜: WT vs F78K 100 Fig. 36 2D-NMR HSQC光譜:WT、F78L vs F78A 101 表 目 錄 TABLE Table 1:硫醇種類 102 Table 2:Chromophore release rate constant k 103 Table 3:本實驗室博士班學長何瑞所提供的螢光動力學數據 103 Scheme 1:產物1的環化機構 104 Scheme 2:產物2的環化機構 105 附錄1:Production of mutant plasmids 106 附錄2:pCAL-n-EK載體:一般結構示意圖 107 附錄3:ApoNCS的基本資料 108 附錄4:質譜質荷比計算分子量 109 附錄5:纯化重組蛋白系統示意圖 110 附錄6:Opening of the binding cleft 111 附錄7:CD spectra of second structure 112 附錄8:Calmodulin affinity column 原理示意圖 113 附錄9:利用UV光譜鑑定活性生色團濃度 114zh_TW
dc.language.isoen_USzh_TW
dc.publisher化學系zh_TW
dc.subjectNeocarzinostatinen_US
dc.subject新抑癌素zh_TW
dc.title新抑癌素苯丙胺酸78突變蛋白對釋放活性生色團的影響zh_TW
dc.titleEffect of Neocarzinostatin Variants of Phe78 Residue on its Chromophore Releaseen_US
dc.typeThesis and Dissertationzh_TW
item.fulltextno fulltext-
item.languageiso639-1en_US-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.grantfulltextnone-
Appears in Collections:化學系所
Show simple item record
 
TAIR Related Article

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.