阿拉伯芥三號澱粉合成酶澱粉吸附區之功能性及結構分析

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研究生：	趙洛芸 Chao, Lo Yun
論文名稱：	阿拉伯芥三號澱粉合成酶澱粉吸附區之功能性及結構分析 Functional and Structural Characterization of Starch Binding Domain in Arabidopsis thaliana Starch Synthase III
指導教授：	張大慈 Chang, Margaret Dah Tsyr
口試委員:	孫玉珠 Sun, Yuh Ju 林彩雲 Lin, Tsai Yun 蘇士哲 Sue, Shih Che
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	100
中文關鍵詞：	阿拉伯芥、澱粉合成酶、澱粉吸附區、合成酵素
外文關鍵詞：	Arabidopsis thaliana, starch synthase, starch binding domain, synthase
相關次數：	點閱：3 下載：0
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澱粉是植物體中主要的能量儲存形式，澱粉合成過程包含三個酵素：腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)、澱粉合成酶(SS)及澱粉分支酶(SBE)，醣類結合模組(CBMs)為能辨認及與醣類專一性結合的蛋白質片段，主要存在於澱粉相關酵素；阿拉伯芥三號澱粉合成酶(AtSSIII)是澱粉合成過程所需的一種酵素，在暫時性澱粉合成中扮演很重要的角色。AtSSIII結構上在N端有一段可能的導引胜肽序列，序列後接著三個澱粉吸附區(SBD)，C端則為催化區(catalytic domain)，阿拉伯芥的澱粉吸附區是第一個在合成酵素中發現的澱粉吸附區，在CAZy資料庫中被分在醣類結合模組中第53個家族，目前研究發現阿拉伯芥三號澱粉合成酶中的三個澱粉吸附區會以串聯形式存在，且其中第二個區域(D2)對其與澱粉的親和性影響最大

因醣類結合模組第53個家族的特性目前了解有限，其結構也尚未被解出，因此本研究希望表現重組蛋白AtSSIIID2後進行功能及結構上的進一步探討。在成功利用大腸桿菌(E. coli)之表現系統表現重組蛋白AtSSIIID2後，利用鎳離子親合層析法(Ni2+-affinity chromatography)純化出此重組蛋白，純化得的蛋白首先利用超高速離心法和液相層析質譜儀分析，發現AtSSIIID2在自然情況下多以單體形式存在，除此之外，實驗發現AtSSIIID2對澱粉的結合親和力在不同pH值的環境中會有所不同，在pH 5的環境中對澱粉親和力最高，而AtSSIIID2與澱粉結合達飽和需七小時以上；另外，利用恆溫滴定熱量計(ITC)測量AtSSIIID2與不同可溶性醣類的結合力，發現AtSSIIID2與短鏈醣的結合力高於長鏈醣，這些實驗結果讓我們對SBD在澱粉合成過程中扮演的角色有更深入了解。

由於醣類結合模組第53個家族的結構仍未知，因此試圖解結構外我們也用軟體進行序列分析及結構預測。序列分析比對後發現AtSSIIID2 和醣類結合模組中其他家族的SBD胺基酸序列相似度極低，但與醣類結合模組中第53個家族的其他成員胺基酸序列相似度很高；結構部分利用Phyre2、SWISS-MODEL及(PS)2三個網路平台作結構預測，其中Phyre2和SWISS-MODEL預測出的結構含8個β-strands，和目前已解出的其他SBD結構組成相同；圓偏光二色光譜(Circular dichroism)分析結果更證明AtSSIIID2結構中含β-strands；AtSSIIID2是第一個在合成酵素發現的SBD，對AtSSIIID2結構的了解或許對其調控有幫助。

中文摘要                                           I
Abstract                                  II
Acknowledgement                                 III
List of Figures                                 VII
List of Tables                                  IX
List of Appendices                           X
Abbreviation                                  XI

Chapter 1  Introduction                           1
1.1  Starch                                   1
1.2  Carbohydrate binding modules (CBMs)       3
1.3  Starch synthase III from Arabidopsis thaliana (AtSSIII)                                   8
1.4  Motivation                                  15

Chapter 2  Materials and Methods          16
2.1  Microbial strains, media and plsmids     16
2.2  Construction of plasmid                  16
2.3  Competent cell preparation                  17
2.4  Transformation                          17
2.5  Small scale expression of AtSSIIID2 in E. coli
                                              17
2.6  Large scale expression of AtSSIIID2 in E. coli
                                              18
2.7  Sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE)                  18
2.8  Western blotting                          19
2.9  Purification of AtSSIIID2 by Ni2+-affinity column chromatography                                  19
2.10  Buffer exchange of AtSSIIID2          20
2.11  Bicinchoninic acid (BCA) assay          20
2.12  Analytical ultracentrifugation          20
2.13  Mass spectrometry determination          21
2.14  Native PAGE                          21
2.15  Effect of pH on AtSSIIID2 binding activity
                                              21
2.16  Isothermal titration calorimetry (ITC)  22
2.17  Circular dichroism (CD)                  22
2.18  Protein homology/analogy recognition engine version 2.0 (Phyre2)                                  22
2.19  Feature-incorporated alignment (FIA)-based homology modeling                                  23
2.20  SWISS-MODEL                          23
2.21  Protein structure prediction server ((PS)2)
                                              24
2.22  Protein crystallization screening          24
2.23  Nuclear magnetic resonance (NMR) heteronuclear single quantum coherence (HSQC) analysis      25
2.24  Sequence analysis tool                  25

Chapter 3  Results                          27
3.1  Construction and small scale expression of recombinant AtSSIIID2                          27
3.2  Purification of recombinant AtSSIIID2 by Ni2+-affinity column chromatography                  28
3.3  Characterization of AtSSIIID2          28
3.4  pH effect on the adsorption of AtSSIIID2 to corn starch                                          29
3.5  Saturation binding assay                  29
3.6  Quantitative measurement of binding affinity between AtSSIIID2 and soluble glycans                  30
3.7  In silico analysis of AtSSIIID2          31
3.8  Secondary structure of AtSSIIID2          32
3.9  Screening for AtSSIIID2 crystallization conditions
                                              33
3.10  Nuclear Magnetic Resonance (NMR)          33

Chapter 4  Discussion                          35
4.1  Characterization of AtSSIIID2          35
4.2  Thermodynamic parameters between AtSSIIID2 and soluble glycans                                  37
4.3  Structure of AtSSIIID2                  38
4.4  Conclusion                                  42

References                                  44
Figures                                          52
Tables                                          82
Appendices                                  92
                                

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