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研究生:蔡謦如
研究生(外文):Ching-Ju Tsai
論文名稱:具細胞毒性之核糖核酸水解酶與四核苷酸複合物之晶體結構研究
論文名稱(外文):Crystal Structure of Cytotoxic RC-RNase and d(ApCpGpA) Complex:
指導教授:孫玉珠
指導教授(外文):Yuh-Ju Sun
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
中文關鍵詞:核糖核酸水解酶四核苷
外文關鍵詞:ribonucleaseRNaseRana catesbeianaonconased(ApCpGpA)tetranucleotideantitumor activitycytotoxicity
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在動物之中,哺乳類動物與牛蛙的RNA水解酶同屬一種家族,但是牛蛙的RNA水解酶除了水解RNA的催化功能外,還有扮演細胞毒性及黏著到癌細胞表面的特性;根據文獻報告,該酵素的細胞毒性與酵素催化功能並無直接關係。為了進一步從結構生物學的角度了解蛙類RNA水解酶的酵素催化機制與細胞毒性的特性,我們從Rana catesbeiana牛蛙取出其中一種RNA水解酶RC-RNase 6,利用共結晶的方法,培養出RC-RNase 6與d(ApCpGpA) 的複合物晶體,希望藉此找到此類蛋白質在催化位置上辨認鹼基的位置,或者是與癌細胞黏著的位置。在所解出2Å的解析度,RC-RNase 6與d(ApCpGpA) 的複合物晶體結構中,我們發現d(ApCpGpA)夾在三個蛋白質分子之間,其中最末兩個核苷酸 (GpA) 緊密的與蛋白質相黏;Thr14, Lys15, Lys16,相鄰的第五號蛋白質分子之Asn54, Asn56, Thr89, Pro105與第七號蛋白質分子上的 Leu58, Arg60, Thr83, Asn84, Thr85, Gly102 都會與d(ApCpGpA) 直接作用。此外,d(ApCpGpA) 所在的位置並非水解RNA的位置,而是在蛋白質的表面,因此我們初步判斷此處可能是黏著到癌細胞表面或是扮演細胞毒性的位置。我們進一步比對蛙類的四種RNA水解酶與牛的兩種RNA水解酶的氨基酸序列,發現Thr14只有在蛙類的酵素中守恆。因此,我們認為Thr14可能與蛙類RNA水解酶的細胞毒性或是辨認癌細胞的特性有關。

A class of bullfrog ribonucleases has been reported to display cytotoxicity and agglutinating activity toward tumor cells. The co-crystallizing crystal structure of a cytotoxic ribonuclease from bullfrog Rana catesbeiana, RC-RNase 6, complexed with d(ApCpGpA) was determined at 2.0 Å resolution. A new binding site was found near T14, K15, K16, F38, Y64, T83 from one protein, and N54, N56, L58, R60, V62, T83, N84, T89, G102 from two neighboring protein molecules. The binding of d(ApCpGpA) did not belong to the productive or non-productive binding near the active site that was occupied by a sulfate ion in this crystal structure. T14, L58, N84, and G102 among the d(ApCpGpA)-interacting residues were found to be conserved in three different ribonucleases from Rana catesbeina and onconase from Rana pipiens via amino acid sequence alignment. From the 2Fo— Fc electron density map, the last two nucleotides of d(ApCpGpA) were well recognized and refined near T14, K15, K16 and Y64 while the first two residues were wobbling and clamped between three protein molecules. This implies the potent lectin activity of T14, K15, K16 and Y64. Among these four residues, only T14 is conserved in the bullfrog RNases. Therefore, d(ApCpGpA) agglutinating site near T14 and the conservation of T14 suggest that T14 might play a crucial role in antitumor activity of the bullfrog ribonucleases.

Chapter 1 Interduction
1.1 Preface
1.2 RNase A
1.3 Ribonuclease Inhibitor
1.4 Onconase 4
1.5 Rana catesbeiana Ribonucleases
1.6 Base Specificity
1.7 Structural Stability
1.8 Cytotoxicity, Antitumor Activity, and Lectin Activity 8
1.9 Protein Crystallization
Chapter 2 Material and Methods
2.1 Sample Preperation
2.2 Protein Concentration Recognition
2.3 Data Collection and Processing
2.4 Crystallization of RC-RNase 4 d(ApCpTpA) complex
2.5 Crystallization of RC-RNase 4 Monisialoganglioside-GM1 complex
2.6 Crystallization of RC-RNase 6 d(ApCpTpA) complex
2.7 Crystallization of RC-RNase 6 d(ApCpGpA) complex
Chapter 3 Result
3.1 Space Group Determination
3.2 Crystal Structural Refinement and Determination
3.3 Protein Structure
3.4 Sulfate Ion Binding Regions
3.5 Nucleic Acid Structure
Chapter 4 Disscussion
4.1 Inhibition of Sulfate Ion at the Active Site
4.2 Another Sulfate Ion Binding Site
4.3 Protein — d(ApCpGpA) Interaction
4.4 d(ApCpGpA) Agglutinating Site on RC-RNase 6 Surface
4.5 Interpretation of antitumor Activity via Specific Residues
Appendix Figures and Tables

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