- 學位論文
開發以α螺旋胜肽鏈建構Type II銅活性中心作為人工氧化酶
Development of α-helical Coiled Coil for the Buildup of Type II Copper Center as an Artificial Oxidase Instead of Oxygenase
摘要
在金屬酵素中，金屬離子於蛋白質中建構活性中心並以高效率及高選擇性催化特定的化學反應。為了了解金屬活性中心結構與反應性間的關聯性，本研究經由胜肽序列的設計(de novo design peptide)，嘗試以bottom up的方式探索人工酵素的建構。本實驗首先利用固相胜肽合成法(SPPS)，合成出一種胜肽鏈， H3a@H4 (G-EIAALKQ-EIAALKK-EHAALKW-EIAALKQ-GYG)，在圓二色光譜(Circular Dichroism Spectrum)的實驗結果發現， H3a@H4完全由α捲曲螺旋的二級結構(α-helix)建構而成，進一步利用分析級超高速離心的分析發現， H3a@H4是三股α-helical coiled coil。除此之外，利用H3a@H4中Histidine的位置。在進一步銅離子滴定實驗中，搭配EPR、ESEEM和EXAFS，發現在胜肽鏈中建構了Cu3:(H3a@H4)3的銅活性中心，配位環境為type II copper center。進一步利用過去實驗室盧培中學長合成的H1a@H4 (G-EHAALKQ-EIAALKK-EIAALKW-EIAALKQ-GYG)與H3a@H4探討有關與ROS間的關聯性，在與雙氧水反應隨時間的變化中，Cu3:(H3a@H4)3與Cu:(H1a@H4)2能有效的降解雙氧水，在與還原劑和CCA反應中Cu3:(H3a@H4)3無明顯•OH或O2-生成，而Cu:(H1a@H4)2有微量的•OH或O2-生成，但仍具有較低的生物毒性，因此利用在生物體之中產氧是未來的方向之一。
關鍵字
胜肽並列摘要
Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Protein design will ultimately allow for the creation of artificial enzymes with novel functions and stability.Various peptide were synthesized by “ Solid Phase Peptide Synthesis ” (SPPS). Successfully, using a de novo designed peptide prepared series of coiled coil peptides (H3a@H4 (G-EIAALKQ-EIAALKK-EHAALKW-EIAALKQ-GYG)). H3a@H4 was helix bundle conformation based on circular dichroism (CD) spectrum. We used Analytical Ultracentrifuge(AUC) to show H3a@H4 was three-stranded coiled coils. H3a@H4 bind Cu(II) with a 1:1 ratio. We used a variety of spectroscopies (EPR, ESSEM, EXAFS) to show Cu3:(H3a@H4)3 was type II copper center, and the coordination geometry of Cu(II), distorted square planar. Further ,We used H3a@H4 and H1a@H4 (G-EHAALKQ-EIAALKK-EIAALKW-EIAALKQ-GYG) synthesized by Lu Pei-Chung to discuss the correlation with ROS. Reacting with hydrogen peroxide, Cu3: (H3a@H4)3 and Cu:(H1a@H4)2 can effectively decompose hydrogen peroxide. Reacting with reducing agent and CCA, Cu3:(H3a@H4)3 has no obvious •OH or O2-, while Cu:( H1a@H4)2 has a small amount of •OH or O2-formation, but it still has low biotoxicity.Then, We can be used Cu3: (H3a@H4)3 and Cu:(H1a@H4)2 in living organisms,like Oxygen production.