Protein Stability Studies of SOX9-HMG Domain

碩士 === 國立清華大學 === 生命科學系 === 89 === SOX9 is DNA-binding protein belongs to a family of HMG-box proteins. SOX9 binds to minor groove of specific DNA sequence (5’-AGAACAATGG-3’) and plays important role as archituture protein in bending the DNA for its biological activity. SOX9 plays an impo...

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Main Author: 楊子瑩
Other Authors: P. C. Lyu
Format: Others
Language:zh-TW
Published: 2001
Online Access:http://ndltd.ncl.edu.tw/handle/43040987685877214631
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spelling ndltd-TW-089NTHU01050232016-01-29T04:33:41Z http://ndltd.ncl.edu.tw/handle/43040987685877214631 Protein Stability Studies of SOX9-HMG Domain SOX9-HMGdomain蛋白質結構穩定性之研究 楊子瑩 碩士 國立清華大學 生命科學系 89 SOX9 is DNA-binding protein belongs to a family of HMG-box proteins. SOX9 binds to minor groove of specific DNA sequence (5’-AGAACAATGG-3’) and plays important role as archituture protein in bending the DNA for its biological activity. SOX9 plays an important role in chondrogenesis as well as in vertebrate sex determination. Theoretial 3D structure of SOX9 is derived using a SRY HMG solution structure solved by NMR. The protein fold of SOX9 consists of three helices constituting a asymmertric L shape. The helices 1 and 2 form one arm of the ‘L shape’ and the third helice form the other arm. In the present work we have studied the influences of various residues of the SOX9 in its stability, folding, and DNA binding properties. Thermal and chemical denarturation methods were used to measure SOX9 stability. The melting of helix of SOX9 was monitored using CD spectroscopic techniques. Alanine-scanning mutagenesis studies revealed that the mutant G40A and N32A are more stable than the wild-type. Replacement of helix breaker (G40A) and charge residues at portion 40 and 32 respectively contributes to the stability. Another mutant H65Y found to increase the stability that the DNA binding decrease to 3 fold. It is interesting to notices that the increase the stability does not directly increase the DNA binding capacity. Phenylanaline at portion 12 and leucine at portion 39 are very important to maintain the fold. Mutation of these residues with F12A and L39A enormously destabilizes the basic fold DNA bind capacity is greatly lost in the F12A mutant. Mutation of other site of SOX9, like P3D, A19Q, P3DA19Q, A11LA58L doesn’t affect the stability or DNA binding capacity of SOX9. 中文摘要..................................................2 第一章:緒論...............................................4 1.1 高移動性群體蛋白質之介紹..............................9 1.1.1 HMG1和HMG1相似之蛋白質.............................10 1.1.2 細胞核、粒線體轉錄因子.............................11 1.1.3 特殊序列結合之蛋白質...............................11 1.2 SOX9的介紹...........................................12 1.2.1 SOX9和軟骨的形成...................................13 1.2.2 SOX9和性別決定.....................................13 1.2.3 HMG-box的結構資訊與DNA結合和彎曲...................14 圖1-1 SRY HMG domain與DNA之複合體........................17 圖1-2 SRY疏水中心一......................................18 圖1-3 SRY疏水中心二......................................19 圖1-4 SRY疏水中心第三....................................20 圖1-5 SOX9 P3D蛋白質.....................................25 圖1-6 SOX9 A19Q蛋白質模擬圖..............................26 圖1-7 SOX9 A11LA58L蛋白質結構............................27 圖1-8 SOX9 Gly 40以及鄰近胺基酸之相關位置................28 圖1-9 SOX9之Phe 12和Leu 39的位置.........................29 圖1-10本實驗突變胺基酸相關位置...........................30 第二章:實驗材料、儀器和方法..............................31 2.1載體(見圖2-1).........................................31 2.2.設計引子( Design primer).............................32 2.3 PCR:突變DNA之合成....................................33 2.4 DNA 洋菜膠體電泳分析(DNA agarose gel)................34 2.5轉殖於細胞(XL-Blue)...................................35 能勝任細胞(Fresh Competent Cell).........................35 化學方法.................................................36 化學轉殖之方法...........................................36 物理方法:electroporation.................................37 electroporation方法轉殖..................................37 2.6.抽出DNA (Minipreparation of Plasmind DNA)............38 2.7質體DNA轉殖到BL21 DE3 component cell...............38 2.8蛋白質的表達與純化....................................38 2.9蛋白質電泳膠分析......................................40 2.10蛋白質濃縮...........................................41 2.11將純化出SOX9用thrombin切S-taq........................42 2.12 高效能液相層析法(HPLC)..............................42 2.13蛋白質分子量 (mass)..................................42 2.14光電比色儀測定濃度...................................42 2.15旋光儀光譜分析.......................................43 2.15.1旋光儀光譜圖(Wavelength scan)......................45 2.15.2熱變性實驗.........................................45 2.15.3化學變性實驗.......................................46 2.15.4蛋白質結構作圖軟體.................................47 2.15.5 Electrophoretic mobility shift assay(EMSA)........48 第三章 結果與討論........................................49 3.1 DNA 洋菜膠體電泳分析.................................49 3.2蛋白質的表達與純化....................................49 3.2.1蛋白質表達之測試....................................49 3.2.2蛋白質的純化........................................49 圖3-1-1 DNA洋菜膠體電泳分析P3D、A19Q之DNA片段............50 圖3-1-2 DNA洋菜膠體電泳分析 P3DA19Q、A11LA58L、F12A、F12Y、F12K、N32A之DNA片段......................................51 圖3-1-3 DNA洋菜膠體電泳分析L39A、G40A、R62A、H65Y的之DNA片段 ................................................52 圖3-2-1 15% SDS PAGE分析蛋白質表現之測試.................53 圖3-2-2 16.5% SDS PAGE分析cell lysate過Native Ni-column之蛋白質 ................................................54 3.2.3 Thrombin之測試.....................................55 3.2.4高效能液相層析法 (HPLC).............................55 圖3-2-3 16.5% SDS PAGE分析Thrombin之測試.................56 圖3-2-4 HPLC.............................................57 3.2.5蛋白質分子量(mass)..................................58 表3-2-5蛋白質的估計值和實驗值之分子量....................58 圖3-2-5蛋白質分子量......................................62 3.3旋光儀光譜分析........................................63 3.3.1 原型SOX9(WT).......................................63 圖3-2A SOX9之熱變性實驗旋光儀光譜圖......................65 圖3-2B SOX9之熱變性實驗旋光儀光譜圖......................66 圖3-3原型SOX9之化學變性實驗..............................67 圖3-4 原型SOX9之ΔGH2O(free energy)......................68 3.3.2 P3D,A19Q,P3DA19Q.................................69 圖3-5 P3D,A19Q,P3DA19Q之旋光儀光譜圖...................70 圖3-6 P3D,A19Q,P3DA19Q之熱變性實驗旋光儀光譜圖.........71 圖3-7 P3D,A19Q,P3DA19Q化學變性實驗.....................72 圖3-8A P3D之ΔGH2O.......................................73 圖3-8B A19Q之ΔGH2O..................................... 74 圖3-8C P3DA19Q之ΔGH2O...................................75 3.3.3 A11LA58L...........................................76 圖3-9 A11LA58L之旋光儀光譜圖與SOX9 WT做比較..............77 圖3-10 A11LA58L之熱變性實驗旋光儀光譜圖..................78 圖3-11A A11LA58L化學變性實驗.............................79 圖3-11B A11LA58L化學變性實驗之ΔGH2O.....................80 3.3.4丙胺酸取代法之突變蛋白..............................81 圖3-12 F12A,N32A,L39A旋光儀光譜圖......................83 圖3-13 G40A,R62A的之旋光儀光譜圖........................84 圖3-14(A) F12A,N32A,L39A,G40A,R62A之熱變性實驗旋光儀光譜圖 ................................................85 圖3-14(B) N32A,G40A,R62A之熱變性實驗旋光儀光譜圖.......86 圖3-15(A) F12A、N32A、L39A、G40A之化學變性實驗...........87 圖3-15(B) N32A、G40A之化學變性實驗.......................88 圖3-16(A) N32A之ΔGH2O...................................89 圖3-16(B) G40A之ΔGH2O...................................90 3.3.5研究Phe 12之穩定結構重要性: F12A,F12Y,F12K........91 圖3-17 F12A,F12Y,F12K之旋光儀光譜圖....................92 圖3-18(A) F12A、F12K、F12Y熱變性實驗.....................93 圖3-18(B) F12Y熱變性實驗.................................94 3.3.6研究疾病H65Y........................................95 圖3-19 H65Y與WT之旋光儀光譜圖比較........................96 圖3-20(A)H65Y之熱變性實驗旋光儀圖........................97 圖3-20(B) H65Y之熱變性實驗旋光儀圖.......................98 3.3.7 DNA結合測試........................................99 圖3-21-1測試各突變蛋白質對S9WT-DNA結合之20% nondenaturing PAGE分析圖.....................................................100 圖3-21-2測試各突變蛋白質對S9WT-DNA結合之20% nondenaturing PAGE分析圖.....................................................101 表一 各突變蛋白質特性分析與原型蛋白質之對照..............102 表二 各突變蛋白質結合DNA測試與原型蛋白質之對照表.........103 第四章 討論..............................................104 圖4-1純化zfSOX9-HMG domain 蛋白質序列....................106 參考文獻.................................................113 P. C. Lyu 呂平江 2001 學位論文 ; thesis 121 zh-TW
collection NDLTD
language zh-TW
format Others
sources NDLTD
author2 P. C. Lyu
author_facet P. C. Lyu
楊子瑩
author 楊子瑩
spellingShingle 楊子瑩
Protein Stability Studies of SOX9-HMG Domain
author_sort 楊子瑩
title Protein Stability Studies of SOX9-HMG Domain
title_short Protein Stability Studies of SOX9-HMG Domain
title_full Protein Stability Studies of SOX9-HMG Domain
title_fullStr Protein Stability Studies of SOX9-HMG Domain
title_full_unstemmed Protein Stability Studies of SOX9-HMG Domain
title_sort protein stability studies of sox9-hmg domain
publishDate 2001
url http://ndltd.ncl.edu.tw/handle/43040987685877214631
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description 碩士 === 國立清華大學 === 生命科學系 === 89 === SOX9 is DNA-binding protein belongs to a family of HMG-box proteins. SOX9 binds to minor groove of specific DNA sequence (5’-AGAACAATGG-3’) and plays important role as archituture protein in bending the DNA for its biological activity. SOX9 plays an important role in chondrogenesis as well as in vertebrate sex determination. Theoretial 3D structure of SOX9 is derived using a SRY HMG solution structure solved by NMR. The protein fold of SOX9 consists of three helices constituting a asymmertric L shape. The helices 1 and 2 form one arm of the ‘L shape’ and the third helice form the other arm. In the present work we have studied the influences of various residues of the SOX9 in its stability, folding, and DNA binding properties. Thermal and chemical denarturation methods were used to measure SOX9 stability. The melting of helix of SOX9 was monitored using CD spectroscopic techniques. Alanine-scanning mutagenesis studies revealed that the mutant G40A and N32A are more stable than the wild-type. Replacement of helix breaker (G40A) and charge residues at portion 40 and 32 respectively contributes to the stability. Another mutant H65Y found to increase the stability that the DNA binding decrease to 3 fold. It is interesting to notices that the increase the stability does not directly increase the DNA binding capacity. Phenylanaline at portion 12 and leucine at portion 39 are very important to maintain the fold. Mutation of these residues with F12A and L39A enormously destabilizes the basic fold DNA bind capacity is greatly lost in the F12A mutant. Mutation of other site of SOX9, like P3D, A19Q, P3DA19Q, A11LA58L doesn’t affect the stability or DNA binding capacity of SOX9. 中文摘要..................................................2 第一章:緒論...............................................4 1.1 高移動性群體蛋白質之介紹..............................9 1.1.1 HMG1和HMG1相似之蛋白質.............................10 1.1.2 細胞核、粒線體轉錄因子.............................11 1.1.3 特殊序列結合之蛋白質...............................11 1.2 SOX9的介紹...........................................12 1.2.1 SOX9和軟骨的形成...................................13 1.2.2 SOX9和性別決定.....................................13 1.2.3 HMG-box的結構資訊與DNA結合和彎曲...................14 圖1-1 SRY HMG domain與DNA之複合體........................17 圖1-2 SRY疏水中心一......................................18 圖1-3 SRY疏水中心二......................................19 圖1-4 SRY疏水中心第三....................................20 圖1-5 SOX9 P3D蛋白質.....................................25 圖1-6 SOX9 A19Q蛋白質模擬圖..............................26 圖1-7 SOX9 A11LA58L蛋白質結構............................27 圖1-8 SOX9 Gly 40以及鄰近胺基酸之相關位置................28 圖1-9 SOX9之Phe 12和Leu 39的位置.........................29 圖1-10本實驗突變胺基酸相關位置...........................30 第二章:實驗材料、儀器和方法..............................31 2.1載體(見圖2-1).........................................31 2.2.設計引子( Design primer).............................32 2.3 PCR:突變DNA之合成....................................33 2.4 DNA 洋菜膠體電泳分析(DNA agarose gel)................34 2.5轉殖於細胞(XL-Blue)...................................35 能勝任細胞(Fresh Competent Cell).........................35 化學方法.................................................36 化學轉殖之方法...........................................36 物理方法:electroporation.................................37 electroporation方法轉殖..................................37 2.6.抽出DNA (Minipreparation of Plasmind DNA)............38 2.7質體DNA轉殖到BL21 DE3 component cell...............38 2.8蛋白質的表達與純化....................................38 2.9蛋白質電泳膠分析......................................40 2.10蛋白質濃縮...........................................41 2.11將純化出SOX9用thrombin切S-taq........................42 2.12 高效能液相層析法(HPLC)..............................42 2.13蛋白質分子量 (mass)..................................42 2.14光電比色儀測定濃度...................................42 2.15旋光儀光譜分析.......................................43 2.15.1旋光儀光譜圖(Wavelength scan)......................45 2.15.2熱變性實驗.........................................45 2.15.3化學變性實驗.......................................46 2.15.4蛋白質結構作圖軟體.................................47 2.15.5 Electrophoretic mobility shift assay(EMSA)........48 第三章 結果與討論........................................49 3.1 DNA 洋菜膠體電泳分析.................................49 3.2蛋白質的表達與純化....................................49 3.2.1蛋白質表達之測試....................................49 3.2.2蛋白質的純化........................................49 圖3-1-1 DNA洋菜膠體電泳分析P3D、A19Q之DNA片段............50 圖3-1-2 DNA洋菜膠體電泳分析 P3DA19Q、A11LA58L、F12A、F12Y、F12K、N32A之DNA片段......................................51 圖3-1-3 DNA洋菜膠體電泳分析L39A、G40A、R62A、H65Y的之DNA片段 ................................................52 圖3-2-1 15% SDS PAGE分析蛋白質表現之測試.................53 圖3-2-2 16.5% SDS PAGE分析cell lysate過Native Ni-column之蛋白質 ................................................54 3.2.3 Thrombin之測試.....................................55 3.2.4高效能液相層析法 (HPLC).............................55 圖3-2-3 16.5% SDS PAGE分析Thrombin之測試.................56 圖3-2-4 HPLC.............................................57 3.2.5蛋白質分子量(mass)..................................58 表3-2-5蛋白質的估計值和實驗值之分子量....................58 圖3-2-5蛋白質分子量......................................62 3.3旋光儀光譜分析........................................63 3.3.1 原型SOX9(WT).......................................63 圖3-2A SOX9之熱變性實驗旋光儀光譜圖......................65 圖3-2B SOX9之熱變性實驗旋光儀光譜圖......................66 圖3-3原型SOX9之化學變性實驗..............................67 圖3-4 原型SOX9之ΔGH2O(free energy)......................68 3.3.2 P3D,A19Q,P3DA19Q.................................69 圖3-5 P3D,A19Q,P3DA19Q之旋光儀光譜圖...................70 圖3-6 P3D,A19Q,P3DA19Q之熱變性實驗旋光儀光譜圖.........71 圖3-7 P3D,A19Q,P3DA19Q化學變性實驗.....................72 圖3-8A P3D之ΔGH2O.......................................73 圖3-8B A19Q之ΔGH2O..................................... 74 圖3-8C P3DA19Q之ΔGH2O...................................75 3.3.3 A11LA58L...........................................76 圖3-9 A11LA58L之旋光儀光譜圖與SOX9 WT做比較..............77 圖3-10 A11LA58L之熱變性實驗旋光儀光譜圖..................78 圖3-11A A11LA58L化學變性實驗.............................79 圖3-11B A11LA58L化學變性實驗之ΔGH2O.....................80 3.3.4丙胺酸取代法之突變蛋白..............................81 圖3-12 F12A,N32A,L39A旋光儀光譜圖......................83 圖3-13 G40A,R62A的之旋光儀光譜圖........................84 圖3-14(A) F12A,N32A,L39A,G40A,R62A之熱變性實驗旋光儀光譜圖 ................................................85 圖3-14(B) N32A,G40A,R62A之熱變性實驗旋光儀光譜圖.......86 圖3-15(A) F12A、N32A、L39A、G40A之化學變性實驗...........87 圖3-15(B) N32A、G40A之化學變性實驗.......................88 圖3-16(A) N32A之ΔGH2O...................................89 圖3-16(B) G40A之ΔGH2O...................................90 3.3.5研究Phe 12之穩定結構重要性: F12A,F12Y,F12K........91 圖3-17 F12A,F12Y,F12K之旋光儀光譜圖....................92 圖3-18(A) F12A、F12K、F12Y熱變性實驗.....................93 圖3-18(B) F12Y熱變性實驗.................................94 3.3.6研究疾病H65Y........................................95 圖3-19 H65Y與WT之旋光儀光譜圖比較........................96 圖3-20(A)H65Y之熱變性實驗旋光儀圖........................97 圖3-20(B) H65Y之熱變性實驗旋光儀圖.......................98 3.3.7 DNA結合測試........................................99 圖3-21-1測試各突變蛋白質對S9WT-DNA結合之20% nondenaturing PAGE分析圖.....................................................100 圖3-21-2測試各突變蛋白質對S9WT-DNA結合之20% nondenaturing PAGE分析圖.....................................................101 表一 各突變蛋白質特性分析與原型蛋白質之對照..............102 表二 各突變蛋白質結合DNA測試與原型蛋白質之對照表.........103 第四章 討論..............................................104 圖4-1純化zfSOX9-HMG domain 蛋白質序列....................106 參考文獻.................................................113