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研究生:許宏嘉
研究生(外文):Hsu. Hung Chia
論文名稱:環狀序列重組應用在具有CSαβ模組的綠豆防禦素第一型之研究
論文名稱(外文):Circular permutation on cysteine- stabilized αβ motif of Vigna radiata plant defensin 1
指導教授:呂平江
指導教授(外文):Lyu. Ping Chiang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:56
中文關鍵詞:環狀序列重組綠豆植物防禦素第一型
外文關鍵詞:VrD1Circular permutationcysteine- stabilized αβ motif
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綠豆植物防禦素第一型 (VrD1),是第一個被研究發現具有抗象蟲活性的植物防禦素,是一個由46個胺基酸組成 (富含半胱胺酸),具有CSαβ motif及四對雙硫鍵來穩定結構的鹼性蛋白質。在我們實驗室之前的研究中,已利用核磁共振光譜儀得到綠豆植物防禦素第一型的水溶液結構;也利用定點突變方法來研究綠豆植物防禦素第一型對麵包蟲α-澱粉水解酶的影響。先前的實驗數據顯示綠豆植物防禦素第一型的第三環型結構以及310螺旋結構在其抑制麵包蟲α-澱粉水解酶的能力上扮演著重要的角色。在此論文中,使用了一項蛋白質工程技術 – 環狀序列重組。這個技術是將蛋白質原有的胺基端和羧基端接在一起,並在另一處產生新的開口,可用來研究蛋白質摺疊、結構穩定度、與蛋白質功能。我們得到兩個環狀序列重組蛋白質VrD1 CP36_G 和 VrD1 CP36_2G,他們都以甲硫胺酸36為新的胺基端,並各自以1個及2個甘胺酸連接舊有的胺基端和羧基端。分析結果發現VrD1 CP36_G 喪失了大部分的結構和抑制澱粉水解酶的功能,然而VrD1 CP36_2G保有了這兩項特性。已知在原有的胺基端和羧基端導入不同長度的鏈結可能會影響環型序列重組蛋白質的折疊,目前的結果顯示導入的鏈結長度必須大於原有胺基端和羧基端之間的距離。儘管VrD1 CP36_2G中新的胺基端和羧基端位於具有酵素抑制功能的第三環型結構上,VrD1 CP36_2G依然具有抑制麵包蟲α-澱粉水解酶的活性。野生型VrD1 及 VrD1 CP36_2G的IC50分別為1.93 μM 和 3.14 μM。因此推論,第三環型結構的主要用途是呈現其上胺基酸殘基所帶的正電荷,用以與麵包蟲α-澱粉水解酶作用進而達到抑制效果。
Mung bean (Vigna radiata) plant defensin 1 (VrD1) is the first reported plant defensin, which exhibits insecticidal activity against Callosobruchus chinensis (bruchid). VrD1 is a 46-residue basic peptide containing a cysteine-stabilized αβ (CSαβ) motif with four disulfide-bonds to stable its structure. Three dimensional structure determined by nuclear magnetic resonance (NMR) spectroscopy and alanine substitutions of noncysteine residues in VrD1 were done in our lab. Our previous studies showed that the loop L3 and the 310 helix played important roles in VrD1 insecticidal function. In this study, a protein engineering method – circular permutation (CP) – was employed on VrD1. The CP rearrangement in a protein is visualized as that the original termini of polypeptide are linked and new termini are created elsewhere. The CP rearranged proteins can be utilized to explore protein folding, stability of structure, and functions. Two CP-VrD1 proteins with residue M36 as the new N-terminus and linkers of one and two glycine (VrD1 CP36_G and VrD1 CP36_2G, respectively) were obtained and investigated. VrD1 CP36_G lost almost all structure and function, while VrD1 CP36_2G kept both characters. The length of linker for native termini may affect the protein folding, and our results revealed that the length of linker should be longer than the distance between the native N- and C-termini. VrD1 CP36_2G inhibited α-amylase although the new termini were created at the functional loop L3 and its IC50 was 1.93 μM while that of wild type VrD1 was 3.14 μM. So the important role of loop L3 may be to display the positively charged residues which participated in the electrostatic interaction between VrD1 and TMA.
中文摘要 1
Abstract 2
Chapter I Introduction 4
1.1 Plant defensins 4
1.2 Vigna radiata plant defensin 1 (VrD1) 5
1.3 Circular permutation (CP) 6
1.4 Aim of this study 7
Chapter II Materials and Methods 8
2.1 Construction of circular-permuted VrD1 (CP-VrD1) plasmids 8
2.2 Expression and purification of VrD1 proteins 9
2.3 Purification and activity assay of Tenebrio molitor α-amylase (TMA) 10
2.4 Protein identification by mass spectrometry 11
2.5 Determination of protein concentration 11
2.6 Assay for inhibitory function of VrD1 proteins against TMA 11
Chapter III Results & Discussion 14
3.1 Overexpression, purification, and identification of VrD1 proteins 14
3.2 Structure Analysis of wt-VrD1 and CP-VrD1 proteins 15
3.3 Inhibitory function of VrD1 proteins against TMA 16
Chapter IV Concusion 18
Reference 51


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