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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊啟伸(Chii-Shen Yang) | |
| dc.contributor.author | Ching-Shin Huang | en |
| dc.contributor.author | 黃慶鑫 | zh_TW |
| dc.date.accessioned | 2021-06-13T15:29:37Z | - |
| dc.date.available | 2010-07-23 | |
| dc.date.copyright | 2008-07-23 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37480 | - |
| dc.description.abstract | 異三元體 G 蛋白質系統是一個被研究相當透徹的主題，它在哺乳類動物和酵母菌的訊息傳遞及生理中扮演了許多重要角色。藉由配體的活化，G 蛋白質耦合性受器 (GPCR) 可促使 Gα 次元產生構形變化，並且引發在 Gα 之中 GTP 置換 GDP。活化狀態的 Gα (Gα-GTP) 會和 Gβγ 次元以及 GPCR 分離，且隨後在細胞內啟發一系列的訊息梯瀑反應。此訊息則會被 Gα 固有的 GTPase 酵素活性給終結，它可水解 GTP 回 GDP 並且將 Gα 恢復回基態。而 Gα 的 GTPase 活性可被 G 蛋白質訊息調控蛋白質 (RGS) 進一步地加速，它會和 Gα 交互作用並且關閉 Gα 的傳訊狀態。因為 GTP 被水解回 GDP，所以 Gα 會再和 Gβγ 重新組合在一起，以等待下一次刺激。
如同哺乳類動物，模式植物阿拉伯芥也擁有異三元體 G 蛋白質系統，且阿拉伯芥異三元體 G 蛋白質的各個成員參與了許多植物的生理功能，如荷爾蒙感受、細胞增殖及逆境抵抗。然而，和哺乳類動物 G 蛋白質廣泛的研究相比較，除了植物異三元體 G蛋白質的生理功能之外，該領域的科學家很少致力於其他方面的研究。因此在本研究之中，我們使用BODIPYTR-GTP 及 Lucifer yellow VS這兩種螢光探針，研究阿拉伯芥 Gα 次元 (AtGPA1) 和 RGS (AtRGS1) 的功能以及它們兩者之間的交互作用。此外，我們也篩選 AtGPA1 以及 AtRGS1 的 RGS 功能區塊的結晶條件。藉由使用 BODIPYTR-GTP，我們可即時監視到 AtRGS1 的 RGS 功能區塊專一地加速 AtGPA1 的 GTPase 活性，並且它與兩蛋白質之間的交互作用是相關聯的。另一個螢光探針 LY 被標定在 AtRGS1 的RGS 功能區塊的三個位置上，展示了在分子層次上兩蛋白質之間的交互作用機制。最後，根據螢光實驗資料，對於 AtGPA1 和 AtRGS1 之間交互作用的關係，我們提出一個兩階段交互作用的模型。 | zh_TW |
| dc.description.abstract | Heterotrimeric G-protein system serves many important roles in signal transduction and physiologies of mammalian and yeast. Upon ligand activation, G-protein coupled receptor (GPCR) could lead to a conformational change in Gα subunit and trigger the replacement of GDP for GTP in Gα. The Gα-GTP, activated form of Gα, dissociates from Gβγ subunit and GPCR, and initializes a series of signal cascading inside the cells. The signal then will be terminated through intrinsic GTPase enzyme activity of Gα which hydrolyzes the bound GTP into GDP and restores Gα to ground state. The GTPase activity of Gα can be further accelerated by a regulator of G-protein signaling (RGS) protein which interacts with Gα and turns off the Gα signaling state. Once GTP is hydrolyzed back to GDP, Gα will reassociate with Gβγ subunit again and ready for next stimulation.
Similar to that of mammalian, Arabidopsis thaliana also harbors heterotrimeric G-protein system and each component of Arabidopsis heterotrimeric G-protein subunits involves many plant physiological functions, such as hormone sensing, cell proliferation and stress resistance. However, compared to the extensive studies on mammalian G proteins, research works on plant heterotrimeric G-protein system seldom focus on aspects other than their physiological functions. In this study, we use two different fluorescent probes, Lucifer yellow vinyl sulfone (LY) and BODIPYTR-GTP, to investigate the functions of Arabidopsis Gα subunit, AtGPA1, and AtRGS1, an Arabidopsis RGS protein, and interactions between them. By using BODIPYTR-GTP, we can real-time monitor protein interaction when RGS box domain of AtRGS1 specifically accelerates GTPase activity of AtGPA1. When another fluorescent probe, LY, was used to label at three different sites of RGS domain of AtRGS1, they can report the interaction and unveil a possible mechanism of these two proteins in molecular level. Finally, we propose a two-step interaction model for AtGPA1 and AtRGS1 based on these fluorescence data. In addition, we also screened crystallization conditions of AtGPA1 and RGS box domain of AtRGS1. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T15:29:37Z (GMT). No. of bitstreams: 1 ntu-97-R95b47204-1.pdf: 2232949 bytes, checksum: 38637f6f6e1e28629de62a278395f31c (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 謝誌 i
Contents ii List of Tables v List of Figures vi 中文摘要 vii Abstract viii Introduction 1 Heterotrimeric G-protein signaling 1 Heterotrimeric G-protein in mammalian 3 Heterotrimeric G-protein in Arabidopsis thaliana 10 Comparison of heterotrimeric G-protein between Arabidopsis and mammalian 14 The purpose of this study 16 Materials and methods 19 Materials 19 Plasmids construction 20 Construction of mutants 20 Expression and purification of AtGPA1 and its mutants 25 Expression and purification of AtRGS1box 27 Expression and purification of AtRGS1domain under denaturing condition 28 Expression and purification of AtRGS1domain and its mutants under native condition 29 Screening of protein crystallization conditions and X-ray diffraction tests 30 Lucifer Yellow labeling and purification 31 Fluorescence assay – AlF4- dependent activation of AtGPA1 32 Fluorescence assay – receptor free GDP/GTP auto-exchange assay 32 Fluorescence assay – GTPase activity of AtGPA1 and GTPase accelerating activity of AtRGS1domain 33 Fluorescence assay – real-time monitoring interaction between AtGPA1 and AtRGS1domain 34 Determination of Lucifer Yellow labeling efficiency on AtRGS1domain mutants interacted with AtGPA1 36 Construction and screening of Pichia Pastoris expression host 36 Results 38 Summary of all constructions and their features 38 Expression and purification of AtGPA1 and its mutants 39 Expression and purification of AtRGS1box 41 Expression and purification of AtRGS1domain and its mutants 43 Expression trials of AtRGS1, AtRGS17TM and AtGCR1 45 Expression screening using Pichia pastoris 47 Screening of protein crystallization conditions and X-ray diffraction tests 48 AlF4- dependent activation of AtGPA1 50 Receptor free GDP/GTP auto-exchange assay 51 Structure and fluorescence properties of fluorescent probes 53 GTPase activity of AtGPA1 and GTPase accelerating activity of AtRGS1domain by BODIPYFL-GTP 55 GTPase activity of AtGPA1 with or without acceleration by AtRGS1domain probed with BODIPYTR-GTP 57 Determination of LY labeling site of native AtRGS1domain 59 Selection of labeling sites 61 Purification of LY-labeled AtRGS1domian proteins 62 GTPase accelerating activities of LY-labeled AtRGS1domian proteins 65 Real-time monitoring interaction between AtGPA1 and AtRGS1domain 66 Mutated sites in AtRGS1domain reveal different labeling efficiency while coupling with AtGPA1 69 Discussions 72 Protein expression in E. coli 72 Protein expression in P. pastoris 73 Crystallization screening of AtGPA1 and AtRGS1domain 74 Biochemical properties of AtGPA1 and its mutants 75 BODIPYTR-GTP based fluorescence assay and biochemical properties of AtRGS1 77 Interaction mechanism between AtGPA1 and AtRGS1 79 Conclusions 82 Perspectives 85 Reference 87 碩士論文口試問與答摘要 96 List of Tables Table 1. Primers used in PCR and point mutations 22 Table 2. Denomination of constructs 24 Table 3. The proteins used in this study 38 Table 4. Summary of protein crystallization screening 49 List of Figures Figure 1. Heterotrimeric G-proetin signaling cycle 2 Figure 2. Structure of G-protein coupled receptors 5 Figure 3. Structure of Heterotrimeric G-protein, effectors, and RGS proteins 9 Figure 4. Schematic framework of this study 18 Figure 5. The chromatography profiles and SDS-PAGE analysis of His6-AtGPA1 40 Figure 6. The chromatography profiles and SDS-PAGE of AtRGS1box-His6 42 Figure 7. The chromatography profiles and SDS-PAGE of AtRGS1domain-His6 44 Figure 8. Expression of AtRGS1, AtRGS17TM and AtGCR1 46 Figure 9. Expression screening of Pichia pastoris 47 Figure 10. AlF4- dependent fluorescence change as a measure of Gα protein activity 51 Figure 11. GDP/GTP auto-exchange activity of AtGPA1 and its mutants 52 Figure 12. Structures of fluorescent probes and their spectra 54 Figure 13. BODIPYFL-GTP as a probe for intrinsic and RGS domain-catalyzed GTPase activity 56 Figure 14. BODIPYTR-GTP as a probe for intrinsic and RGS domain-catalyzed GTPase activity 58 Figure 15. LY labeling site analysis of native AtRGS1domain 60 Figure 16. Strategy of mutation sites selection 61 Figure 17. Purification of LY-labeled AtRGS1domian(DC) and AtRGS1domain(EDC) 63 Figure 18. Purification of AtRGS1domian(SC1) and AtRGS1domain(ESC1) 64 Figure 19. Purification of AtRGS1domian(SC2) 65 Figure 20. BODIPYTR-GTP/GDP fluorescence change is still accelerated by LY-labeled AtRGS1domain 66 Figure 21. Different sites labeled with LY show different fluorescence features on protein-protein interaction 68 Figure 22. Labeling efficiency analyses of AtRGS1domain mutants 71 Figure 23. Illustration of a two-step interaction mechanism 81 | |
| dc.language.iso | en | |
| dc.subject | G 蛋白質訊息調節蛋白質 | zh_TW |
| dc.subject | 訊息傳遞 | zh_TW |
| dc.subject | G 蛋白質 | zh_TW |
| dc.subject | 阿拉伯芥 | zh_TW |
| dc.subject | 分子機制 | zh_TW |
| dc.subject | 螢光 | zh_TW |
| dc.subject | 異三元體 G 蛋白質 | zh_TW |
| dc.subject | AtRGS1 | en |
| dc.subject | G-protein | en |
| dc.subject | signal transduction | en |
| dc.subject | Arabidopsis thaliana | en |
| dc.subject | AtGPA1 | en |
| dc.subject | heterotrimeric G-protein | en |
| dc.subject | molecular mechanism | en |
| dc.subject | fluorescence | en |
| dc.title | 阿拉伯芥 G 蛋白質訊息傳遞系統和 G 蛋白質訊息調節蛋白質 (AtRGS1) 分子機制探討 | zh_TW |
| dc.title | Molecular Mechanism of Arabidopsis thaliana G-protein System and Its Signaling Regulatory Protein, AtRGS1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李平篤(Ping-Du Lee),梁博煌(Po-Huang Liang),楊健志(Chien-Chih Yang),陳俊任(Chun-Jen Chen) | |
| dc.subject.keyword | 阿拉伯芥,G 蛋白質,訊息傳遞,G 蛋白質訊息調節蛋白質,異三元體 G 蛋白質,分子機制,螢光, | zh_TW |
| dc.subject.keyword | Arabidopsis thaliana,G-protein,signal transduction,AtRGS1,AtGPA1,heterotrimeric G-protein,molecular mechanism,fluorescence, | en |
| dc.relation.page | 96 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-07-16 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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