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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 常怡雍(Yee-yung Charng) | |
| dc.contributor.author | Zih-teng Chen | en |
| dc.contributor.author | 陳子騰 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:01:45Z | - |
| dc.date.copyright | 2013-08-27 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17223 | - |
| dc.description.abstract | Prokaryotic GrpE acts as a nucleotide-exchange factor in the Hsp70/DnaK chaperone machinery. Two GrpE homologs were identified in Arabidopsis mitochondria (Mge1 and Mge2). Mge2, but not Mge1, restores the growth of heat-sensitive E. coli grpE mutant, DA16, at 43 oC. Although Mge1 and Mge2 are highly similar in primary structure, Mge2 possesses an extra peptide derived from a retained in-frame intron sequence. Phenotyping of Mge2 T-DNA knockout lines reveals that Mge2 is specifically required for tolerating prolonged exposure to moderately high temperature. To characterize how Mge2 confers tolerance to high temperature, plasmids expressing recombinant Mge proteins were constructed and used for complementary assay. Recombinant Mge proteins were purified and analyzed by circular dichroism. The results suggested Mge2 was more thermostable than Mge1. And the in-frame intron-derived peptide sequence of Mge2 might not affect the thermostability in vitro. Furthermore, analysis of domain-swapped Mge proteins indicated that both the long α-helix domain and the β-sheet domain might affect the thermostability of Mge proteins, but the long α-helix domain might be predominant in affecting the thermostability. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:01:45Z (GMT). No. of bitstreams: 1 ntu-102-R00b22024-1.pdf: 2756517 bytes, checksum: 56cc4d2eebcc08ea32010262cec84ced (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | Contents i
Abbreviation iv Abstract v 中文摘要 vi Figure contents vii Table contents viii Chapter I. Introduction 1 1.1 Heat stress 1 1.2 Heat shock proteins and Hsp70s 1 1.3 Mechanism of KJE chaperone cycle 2 1.4 Structure and thermodynamic properties of GrpE protein 4 1.5 GrpE homologs in eukaryotes 9 1.6 Arabidopsis Mge1 and Mge2 might be specialized for different stress 10 1.7 Objective of this study 12 Chapter II. Materials and Methods 14 2.1 Cloning of Arabidopsis Mge genes 14 2.1.1 Plasmids construction for production of Strep-tagged recombinant Mge1 and Mge2 proteins 14 2.1.2 Plasmid construction for production of recombinant Strep-tagged Mge2Δ protein 15 2.1.3 Plasmid construction for production of Strep-tagged domain-swapped Mge proteins 15 2.2 Complementary assay 18 2.3 Purification of Mge proteins 19 2.3.1 Protein expression 19 2.3.2 Cell lysis 20 2.3.3 Affinity chromatography 20 2.3.4 Ion-Exchange chromatography 21 2.3.5 Gel-filtration chromatography 22 2.4 Circular dichorism (CD) 22 2.5 Mass analysis 23 2.5.1 In-gel digestion 23 2.5.2 Zip-Tip purification 24 2.5.3 MS analysis 24 Chapter III. Results 26 3.1 The Strep-tag does not alter the function of recombinant Mge proteins. 26 3.2 Purification of recombinant Strep-tagged Mge proteins. 28 3.3 CD analysis showed that Mge2 was more thermostable than Mge1 at elevated temperatures. 29 3.4 Deletion of the intron-derived peptide sequence in Mge2 did not affect the function of Mge2 in complementation of E. coli DA16 mutant. 31 3.5 Deletion of the intron-derived peptide sequence in Mge2 did not affect protein thermostability in vitro. 32 3.6 The long α-helix domain in Mge determines the capacity in conferring tolerance to high temperature in vivo. 33 3.7 CD analysis showed that the long α-helix is the most important domain affecting Mge thermostability in vitro. 35 Chapter IV. Discussion 37 4.1 Mge1 and Mge2 might possess different affinity to DnaK. 37 4.2 The Arabidopsis Mge proteins might function similarly to S. cerevisiae Mge1p rather than E. coli GrpE. 38 4.3 Mge2 might remain active during chronic heat stress. 40 4.4 The long α-helix domain might be predominant to the thermostability of Arabidopsis Mge proteins. 41 Chapter V. Future work 44 5.1 complementation analysis of Mge2Δ in mge2 mutant plants. 44 5.2 Identification of the relationship between the thermal unfolding transition and monomerization of Mge proteins 44 5.3 Measurements of NEF activity of Mge proteins 45 References 46 Figures 52 Tables 68 Appendix 73 碩士論文口試問答摘要 83 | |
| dc.language.iso | en | |
| dc.title | 阿拉伯芥粒線體GrpE蛋白質之熱穩定性研究 | zh_TW |
| dc.title | Thermostability Analysis of Two Arabidopsis Mitochondrial GrpE Proteins | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 楊健志(Chien-chih Yang) | |
| dc.contributor.oralexamcommittee | 王愛玉,陳佩燁,黃楓婷 | |
| dc.subject.keyword | 阿拉伯芥,GrpE,Mge,熱穩定性, | zh_TW |
| dc.subject.keyword | Arabidopsis,GrpE,Mge,thermostability, | en |
| dc.relation.page | 87 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2013-08-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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