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研究生:張凱棣
研究生(外文):KAI-TI CHANG
論文名稱:非典型肺炎3CL蛋白酶C145A突變蛋白受質結合態的結晶結構:提供以結構基礎藥物設計的線索
論文名稱(外文):Crystal structure of SARS 3CL mutant C145A in a product-bound state: implication for the design of structure-based inhibitors
指導教授:王惠鈞
指導教授(外文):Andrew H.-J. Wang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:92
語文別:英文
論文頁數:57
中文關鍵詞:結晶非典型肺炎冠狀病毒結構
外文關鍵詞:3C-like proteinasemain proteinasecrystal structuresevere acute repiratory syndromcoronavirusSARS3CL
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  一種新型的冠狀病毒(SARS-CoV)是造成嚴重急性呼吸道症候群
爆發的原因。這個冠狀病毒利用SARS 3C-like proteinase 做大範圍的
酵素分解反應來產生複製和轉譯所需要的酵素。因為這個原因SARS
3C-like proteinase 就成為藥物設計的主要目標。把在SARS 3C-like
proteinase 中,參與催化作用的一個殘基,位於第145 的半氨胱酸
cysteine),突變成丙胺酸(Alanine)。這個突變的酵素依序被大量表
現、純化和結晶。得到一個單斜晶系、空間群為C2 和解析度2.5 Å
的晶體。每個不對稱單元(asymmetric unit)含有一個二聚體。由結構知
道活性區域被屬於另一個不稱單元的單體所鑲嵌。這個研究提供一些
資訊來了解基質和酵素結合,能夠幫助針對嚴重呼吸道症候群或是其
他相關冠狀病毒的藥物設計。
A newly identified severe acute respiratory syndrome coronavirus
(SARS-CoV) is the etiological agent responsible for the outbreak of
severe acute respiratory syndrome (SARS). The 3C-like proteinase (also
called main proteinase) is a key enzyme for mediating viral replication
and transcription through extensive proteolytic processing and an
essential target for drug development. One of the catalytic residues of
SARS 3C-like protease, Cys145, was replaced with Ala and the C145A
mutant was over-expressed, purified and crystallized. The plate-like
crystal diffracts to 2.5Å and belongs to C2 space group. There is one
dimer in an asymmetric unit. The structure revealed that the active site
region is penetrated by another monomer which belongs to another
asymmetric unit. This study provides a basic understanding of the
substrate binding, which is helpful for the design of structure-based
inhibitors SARS and related coronaviruses.
ACKNOWLEDGEMENTS........................................................................ I
ABSTRACT............................................................................................... II
LIST OF FIGURES ................................................................................. VI
LIST OF TABLES .................................................................................. VII
CHAPTER 1 INTRODUCTION.............................................................1
1.1 Severe acute respiratory syndorme (SARS) ..........................1
1.2 Coronaviridae.........................................................................2
1.3 SARS-CoV ...........................................................................2
1.4 3C-like proteinase (3CL proteinase)......................................3
1.5 The structure of 3CLproteinase .............................................4
1.6 X-ray crystallography ............................................................6
1.7 The aim of this study .............................................................7
CHAPTER 2 MATERIALS AND METHODS.....................................9
2.1 Materials ................................................................................9
2.2 Preparation of circular plasmid............................................10
2.3 Polymerase chain reaction (PCR)........................................10
2.4 Transformation of competent E. coli cells ..........................11
2.5 Preparation of competent E. coli cells .................................12
2.6 Site-directed mutagenesis (STRATAGENE #200518).......13
2.6.1 Primer design................................................................13
2.6.2 Mutant strand synthesis reaction (Thermal cycling)....14
2.6.3 Dpn I digestion .............................................................14
2.6.4 Transformation .............................................................14
2.7 Over-expression of GST-3CL proteinase (C145A).............15
2.8 Purification of 3CL proteinase (C145A) .............................15
2.9 Enzyme activity assay..........................................................16
2.10 Crystallization....................................................................17
2.11 Soaking ..............................................................................17
2.12 Data Collection and processing .........................................18
2.13 Structure determination and refinement ............................18
CHAPTER 3 RESULTS ......................................................................20
3.1 Cloning, over-expression and purification of GST-3CL
proteinase (C145A)...........................................................20
3.2 Enzyme activity assay..........................................................21
3.3 X-ray crystallography ..........................................................22
3.3.1 Crystallization and soaking ..........................................22
3.3.2 Data collection..............................................................23
3.3.3 Structure determination ................................................23
3.4 Crystal structure of 3CL proteinase (C145A) .....................24
3.4.1 Overall structure ...........................................................24
3.4.2 Active site.....................................................................24
3.4.3 Product-bond state........................................................25
3.4.4 S1 subsite......................................................................25
3.4.5 S2 subsite......................................................................26
3.4.6 S3 subsite......................................................................26
3.4.7 Other subsite.................................................................26
CHAPTER 4 DISCUSSION..................................................................27
CHAPTER 5 REFERENCE ..................................................................55
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