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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38695完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 吳世雄(Shih-Hsiung Wu) | |
| dc.contributor.author | Hui-Jou Chou | en |
| dc.contributor.author | 周慧柔 | zh_TW |
| dc.date.accessioned | 2021-06-13T16:42:24Z | - |
| dc.date.available | 2013-08-16 | |
| dc.date.copyright | 2011-08-16 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-17 | |
| dc.identifier.citation | 1. Swamy, K.H. and Goldberg, A.L. (1981) E. coli contains eight soluble proteolytic activities, one being ATP dependent. Nature, 292, 652-654.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38695 | - |
| dc.description.abstract | ATP-dependent Lon蛋白酶是維持蛋白質功能與結構之衡定的重要成員之一，普遍分布在各種生物體中。在功能方面，Lon能夠分解細胞內不正常累積的蛋白質以及特定的調節蛋白。Lon蛋白酶可大致分為兩種亞型，LonA和LonB。相較於LonB，LonA已廣泛的被學者們研究。先前的研究已經指出極端嗜熱古生菌(Thermococcus kodakaraensis KOD1)的Lon蛋白酶由N端ATP水解酶功能區及C端蛋白水解酶功能區所組成，在宿主體內是以膜蛋白的形式存在，屬於LonB。在本論文中，藉由刪去TK-Lon的穿膜區域，我們設計了1個TK-Lon的突變株蛋白質(TK-LonΔTM)，並探討此突變株蛋白質的功能與結構特性。在大量表現並純化出TK-LonΔTM後，實驗證明此突變株蛋白酶具有ATP水解酶與蛋白水解酶活性。利用電泳凝膠位移測定(EMSA)，我們發現TK-LonΔTM具有DNA-binding的活性。在伴護功能活性測定實驗中，我們觀察到不論是在加熱誘導或是利用化學試劑破壞使蛋白質不規則聚集的情況下，TK-LonΔTM都能預防變性蛋白質的不規則聚集。在熱穩定性的研究中，利用示差掃描熱分析儀(DSC)可觀察到TK-LonΔTM的熔點(Tm)為98.9℃，我們認為TK-LonΔTM仍然保留原來的極端熱穩定性。在結構方面，遠紫外光圓二色偏光光譜圖顯示TK-LonΔTM以α螺旋為主要二級結構；近紫外光圓二色偏光光譜圖顯示TK-LonΔTM擁有完整的三級結構。進一步利用凝膠過濾層析法、分析級超高速離心儀以及穿透式電子顯微鏡分析TK-LonΔTM的四級結構，我們的結果都顯示TK-LonΔTM會聚集形成六聚體(hexamer)，我們認為此突變株與野生型的結構極為相似。這些實驗結果都可以證明Lon蛋白酶的功能與結構特性不只存在於LonA中，也高度保留在LonB中。 | zh_TW |
| dc.description.abstract | ATP-dependent Lon proteases degrade specific short-lived regulatory proteins and are key components of the protein quality control systems in the cell, which are universally distributed in all kingdoms of life. Lon protease can be divided into two subfamilies, LonA and LonB. LonA is well-studied as compared with LonB. Previous studies have shown that Lon protease from Thermococcus kodakaraensis KOD1 (TK-Lon) which belong to LonB is composed of an N-terminal ATPase domain and a C-terminal protease domain and is a membrane-bound protein in its native host. In this study, we designed a TK-Lon mutant protein (TK-LonΔTM) with a deletion of the membrane-anchoring region and characterized its function and structure. TK-LonΔTM was overexpressed in E.coli and purified from soluble fraction displaying ATPase and proteolytic activity. Electrophoresis mobility shift assay showed that TK-LonΔTM has DNA-binding activity. Chaperone activity assay indicated that TK-LonΔTM can prevent aggregation of denature proteins under thermal stress or chemical stress. The melting temperature of TK-LonΔTM was observed at 98.9 ℃ by differential scanning calorimetry, suggesting the extreme thermostable of TK-LonΔTM. Far-UV CD and near-UV CD measurements revealed that TK-LonΔTM consists of α-helices as the major secondary structure and possesses well-defined three-dimensional structure, respectively. Our gel-filtration chromatography assay, analytical ultracentrifugation and transmission electron microscopy all displayed that TK-LonΔTM assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. These findings showed that function and structure of Lon protease are conserved in the LonA and LonB subfamilies. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T16:42:24Z (GMT). No. of bitstreams: 1 ntu-100-R98B46027-1.pdf: 2491183 bytes, checksum: 9c4d86009f6c135f20401a8bad76e6b1 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | Table of contents
謝誌 ............................................................................................................................ IV 中文摘要 ...................................................................................................................... V Abstract ...................................................................................................................... VI Abbreviation table ..................................................................................................... VIII Chapter 1 Introduction ................................................................................................... 1 1-1 The role of Lon protease in organism ................................................................ 1 1-2 Classification of Lon protease ............................................................................ 2 1-3 The function of Lon protease ............................................................................. 3 1-4 The structure of Lon protease ............................................................................. 5 1-5 Hyperthermophilic archaeon .............................................................................. 5 1-6 The goal of this study ......................................................................................... 7 Chapter 2 Materials and Methods .................................................................................. 9 2.1 Sequence analysis ............................................................................................... 9 2.2 Cloning ............................................................................................................... 9 2.3 Expression and Purification .............................................................................. 10 2.4 Protease Assay .................................................................................................. 11 2.5 Peptidase Assay ................................................................................................ 11 2.6 ATPase Assay .................................................................................................... 12 2.7 Electrophoresis Mobility Shift Assay ............................................................... 13 2.8 Chaperone Activity Assay ................................................................................ 14 2.8.1 Under thermal stress .............................................................................. 14 2.8.2 Under chemical stress ............................................................................ 14 2.9 Circular Dichroism (CD) spectra ...................................................................... 15 2.10 Analytical Ultracentrifugation (AUC) ............................................................ 16 2.11 Transmission Electron Microscopy (TEM) .................................................... 17 2.12 Differential Scanning Calorimetry (DSC) ...................................................... 17 2.13 Analytical Gel Filtration Assay ...................................................................... 18 2.14 Surface hydrophobicity .................................................................................. 18 2.15 Autolysis of TK-LonΔTM .............................................................................. 18 2.16 Homology modeling of TK-LonΔTM hexamer structure .............................. 19 Chapter 3 Results ...................................................................................................... 20 3-1 Genome analysis of T. kodakaraensis KOD1 Lon protease ............................. 20 3-2 Expression and purification .............................................................................. 21 3-3 Characterization of proteolytic and ATPase activities of TK-LonΔTM ........... 22 3-4 Characterization of chaperone-like activity of TK-LonΔTM .......................... 23 3-5 Characterization of DNA-binding activity of TK-LonΔTM ............................ 25 3-6 Thermal stability .............................................................................................. 26 3-7 Secondary and tertiary structure ....................................................................... 27 3-8 Oligomerization state of TK-LonΔTM ............................................................ 28 Chapter 4 Discussions ................................................................................................. 30 4-1 Phylogenetic analysis of TK-Lon ..................................................................... 30 4-2 The discrepancy between TK-Lon and TK-LonΔTM for protease and ATPase activity in optimum temperature ............................................................................ 31 4-3 DNA binding and α domain ............................................................................. 32 4-4 Characterization of chaperone-like activity of TK-Lon ................................... 33 4-5 Thermal stability .............................................................................................. 35 4-6 Nature of TK-Lon oligomerization .................................................................. 37 4-7 Physiological roles of multi-functional TK-Lon .............................................. 39 Figures ..................................................................................................................... 40 Figure 1. Domain structures of TK-Lon protease and its mutant design................ 40 Figure 2. Multiple alignments of amino acid sequences of TK-Lon and other LonA proteases ................................................................................................................. 41 Figure 3. Multiple alignments of amino acid sequences of TK-Lon and other LonB proteases ................................................................................................................. 42 Figure 4. SDS-PAGE of purified recombinant TK-Lon ΔTM and TK-LonP ......... 43 Figure 5. Autolysis of TK-Lon ΔTM ...................................................................... 44 Figure 6. Effects of temperature on protease and ATPase activities of TK-Lon ΔTM ........................................................................................................................... 45 Figure 7. Effects of temperature on peptidase activities of TK-Lon ΔTM ............. 46 Figure 8. effect of ATP on protease activities of TKLonΔTM ............................... 47 Figure 9. Chaperone-like activity of TK-LonΔTM under thermal stress ............... 48 Figure 10. Chaperone-like activity of TK-LonΔTM under chemical stress ........... 49 Figure 11. Electrophoretic mobility shift assay ...................................................... 50 Figure 12. Thermostability of TK-LonΔTM measured by CD .............................. 51 Figure 13. Thermal denaturation of TK-Lon ΔTM by differential scanning calorimetry .............................................................................................................. 52 Figure 14. Hydrophobicity measurement ............................................................... 53 Figure 15. Far-ultraviolet circular dichroism spectra ............................................. 54 Figure 16. Near-ultraviolet circular dichroism spectra ........................................... 55 Figure 17. Estimation of the molecular mass of native TK-LonΔTM by analytical gel filtration ............................................................................................................ 56 Figure 18. Sedimentation velocity experiment of TK-LonΔTM ............................ 57 Figure 19. Transmission electron microscopy images of TK-LonΔTM ................ 58 Figure 20. Phylogenetic analysis of Lon protease ( by Jotun Hein Method) ......... 59 Figure 21. The amino acid alignment of α-domains ............................................... 60 Figure 22. Stereo view of homology model TK-LonΔTM ..................................... 62 Tables ....................................................................................................................... 63 Table 1. Strains, Plasmids and Oligonucleotides used in this study ....................... 63 Table 2. Buffer composition used in this study ...................................................... 64 Table 3. The secondary structure ratios of TK-LonΔTM and TK-LonP ................ 65 Table 4. Statistical comparison of the hyperthermophilic, thermophilic and mesophilic Lon proteases ....................................................................................... 66 Table 5. Statistical value of TK-LonΔTM interface interaction ............................. 67 References ................................................................................................................. 68 | |
| dc.language.iso | en | |
| dc.subject | 六聚體 | zh_TW |
| dc.subject | LonB | zh_TW |
| dc.subject | 伴護 | zh_TW |
| dc.subject | 極端嗜熱古生菌 | zh_TW |
| dc.subject | Lon 蛋白酶 | zh_TW |
| dc.subject | Hexamer | en |
| dc.subject | Chaperone | en |
| dc.subject | LonB | en |
| dc.subject | Thermococcus kodakaraensis KOD1 | en |
| dc.subject | Lon protease | en |
| dc.title | 極端嗜熱古生菌Thermococcus kodakaraensis Lon蛋白酶功能與結構之研究 | zh_TW |
| dc.title | Function-Structural Analysis of Lon Protease from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳金榜(Chinpan Chen),徐駿森(Chun-Hua Hsu),李岳倫(Alan Yueh-Luen Lee) | |
| dc.subject.keyword | Lon 蛋白酶,極端嗜熱古生菌,LonB,伴護,六聚體, | zh_TW |
| dc.subject.keyword | Lon protease,Thermococcus kodakaraensis KOD1,LonB,Chaperone,Hexamer, | en |
| dc.relation.page | 75 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-07-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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