臺灣博碩士論文加值系統

Lon 蛋白酵素高度保留存在於各種生物體中，且為多功能的單一聚合型酵素。先前的研究指出 Lon 蛋白酵素具有 ATPase、Protease、Peptidase、Chaperone、DNA-binding 等生物活性，透過其多功能的特性，Lon 蛋白酵素 可以維持生物體內蛋白質完整的功能與結構，或適時降解目標蛋白，進而調控體內蛋白質質量的恆定。本論文選擇臺灣本土烏來地區所分離出的嗜熱菌 Meiothermus taiwanensis (WR-220)，選殖出其基因體內被推論為 Lon 蛋白酵素的三個基因，透過表現與純化，以此三個蛋白酵素 (Mt-LonA1、Mt-LonA2、Mt-TTC1975) 為研究對象，探討此三個蛋白酵素在結構與功能的特徵，進而比較此三個蛋白酵素的差異。
Lon 蛋白酵素分為 A-type 與 B-type，其中 A-type Lon 具有三個功能區(domain): N 端功能區、ATP 水解酵素功能區以及 C 端蛋白水解酵素功能區。透過應用軟體分析此三個蛋白酵素的一級結構，Mt-LonA1 與 Mt-LonA2 高度保留 A-type Lon 的三個功能區，而 Mt-TTC1975，只保留 C 端蛋白水解酵素功能區，因此我們認為 Mt-TTC1975 不應歸類為 Lon 蛋白酵素。
在結構方面，利用原二色偏光儀分析 Mt-LonA1、Mt-LonA2 與 Mt-TTC1975，結果顯示此三個蛋白酵素皆以 α-螺旋為主要的二級結構，並具有完整的三級結構。進一步利用分析級超高速離心儀、原態膠體電泳以及電子顯微鏡分析此三個蛋白酵素的四級結構，我們認為 Mt-LonA1 以六聚合體 (hexamer) 為主，Mt-LonA2 以二聚合體 (dimer) 與五聚合體 (pentamer) 為主，而 Mt-TTC1975 以六聚合體 (hexamer) 與七聚合體 (heptamer) 為主。
活性方面，Mt-LonA1 具有 ATPase、Protease、Peptidase、Chaperone 等活性，而 Mt-LonA2 具有 ATPase、Protease、Peptidase、DNA-binding 等活性，實驗結果顯示此兩個蛋白酶為 A-type Lon 蛋白酵素，值得注意的是，Mt-LonA1 缺乏DNA-binding 的活性，而 Mt-LonA2 缺乏 chaperone 的活性，我們認為 Mt-LonA1 與 Mt-LonA2 在生物體內所扮演的角色可能不盡相同。而 Mt-TTC1975 則只擁有 chaperone 活性，此結果也與 Mt-TTC1975 不應歸類為 Lon 蛋白酵素的推論相符。
最後，我們討論 Mt-LonA1 與 Mt-LonA2 在 DNA-binding 活性的差異，藉由分析此兩個蛋白酵素與已被清楚研究的 Bt-Lon 之 α-domain 一級結構，以及比較 α-domain 的分子模型，我們認為 Mt-LonA2 之 K527 胺基酸與一段的保留序列 K-K-R ，可能為提供與 DNA 結合所需之正電荷的重要胺基酸。

The Lon proteases had been known as one of the most evolutionarily conserved proteins. According to previous findings, Lon proteases possessed ATPase, protease, peptidase and chaperone activities. Based on these multi-functional Lon proteases, the protein quality control system and the regulation of metabolic process could both work well. In this study, the function-structural characterizations among Mt-LonA1, Mt-LonA2 and Mt-TTC1975 from Meiothermus taiwanensis would be juxtaposed to express the contrast.
The Lon protease family could be divided into two subfamilies, LonA and LonB, mainly based on the sources and the domain structures of these proteins. The LonA consisted of a variable N-terminal domain (N domain), a central ATPase domain (A domain), and a C-terminal protease domain (P domain) on a single polypeptide. Depending on the analysis of primary structures of Mt-LonA1, Mt-LonA2 and Mt-TTC1975, This study considered that Mt-LonA1 and Mt-LonA2 both should be classified as the LonA subfamily, for Mt-LonA1 and Mt-LonA2 both possessed the classical LonA-type domains. For Mt-TTC1975, it only possessed a high similarity in protease domain with canonical LonA. Therefore, it should not be classified as the Lon protease.
Structural characteristic results by circular dichroism showed that Mt-LonA1, Mt-LonA2 and Mt-TTC1975 possessed mostly α-helical secondary structures and they all possesed well-defined three-dimensional structures. For quaternary structures, the AUC data, Native-PAGE and EM graph revealed that Mt-LonA1 functions mainly as a hexamer; the AUC data and Native-PAGE revealed that Mt-LonA2 might function as a mixture of dimer and pentamer; Native-PAGE revealed that Mt-TTC1975 functions as a hexamer or a heptamer.
Functional characteristic results showed that Mt-LonA1 exhibited the ATPase, protease, peptidase and chaperone activity; Mt-LonA2 exhibited the ATPase, protease, peptidase and DNA-binding activity; Mt-TTC1975 exhibited the chaperone activity only.
Lastly, the comparison of primary structure of α-domains and the results of homology modeling suggested that the K527 residue and the K-K-R conserved region of Mt-LonA2 might critically influence the DNA-binding activity.

Table of Contents
口試委員審定......................i
謝誌....................ii
中文摘要....................iii
Abstract....................v
Abbreviation Table ......vii
1. Introduction......1
1-1. Thermophiles......1
1-2. Lon protease......2
Figure 1-1. Domain structures of LonA and LonB subfamily..8
Figure 1-2. Schematic diagram of the functions of Lon....9
2. Materials and Methods....10
2-1. Materials.....10
2-2. Methods......12
3. Results......22
PART 1: Analysis of the three putative Lon proteases from Meiothermus taiwanensis WR-220......22
PART 2: Structural characteristic and thermal stability of Mt-LonA1, Mt-LonA2 and Mt-TTC1975......24
PART 3: Functional characteristics of Mt-LonA1, Mt-LonA2 and Mt-TTC1975..28
4. Discussion......31
4-1. Phylogenetic analysis of Mt-LonA1, Mt-LonA2 and Mt-TTC1975..31
4-2. Thermal stability of Mt-LonA1, Mt-LonA2 and Mt-TTC1975...32
4-3. Structural characteristics of Mt-LonA1, Mt-LonA2 and Mt-TTC1975..33
4-4. Catalytic activity of Mt-LonA1, Mt-LonA2 and Mt-TTC1975...34
4-5. Characterization of chaperone activity of Mt-LonA1, Mt-LonA2 and Mt-TTC1975......35
4-6. DNA-binding and α-domain....36
5. Figure......38
Figure 5-1. Putative promoter region of Mt-LonA1 and Mt-LonA2..38
Figure 5-2. The amino acid sequence alignment of Mt-LonA1 and other Lon protease
.......39
Figure 5-3. The amino acid sequence alignment of Mt-LonA2 and other Lon protease
.......40
Figure 5-4. The amino acid sequence alignment of Mt-LonA1 and other Mt-LonA2
.......41
Figure 5-5. The phylogenetic tree of Lon proteases...42
Figure 5-6. SDS-PAGE of purified recombinant Mt-LonA1, Mt-LonA2 and Mt-TTC1975......43
Figure 5-7. Far-UV CD spectra......44
Figure 5-8. Near-UV CD spectra....45
Figure 5-9. Thermostability of Mt-LonA1, Mt-LonA2 and Mt-TTC1975...46
Figure 5-10. Sedimentation velocity experiment of Mt-LonA1...47
Figure 5-11. Sedimentation velocity experiment of Mt-LonA2...48
Figure 5-12. Native PAGE of Mt-LonA1, Mt-LonA2 and Mt-TTC1975..49
Figure 5-13. Transmission electron microscopy images of Mt-LonA1..50
Figure 5-14. ATPase activity assay.....51
Figure 5-15. Protease activity assay....52
Figure 5-16. Peptidase activity assay....53
Figure 5-17. Chaperone activity assay...54
Figure 5-18. Electrophoretic mobility shift assay....55
Figure 5-19. Amino acid sequence alignment of α-domains..56
Figure 5-20. Homology modeling of α-domains...57
6. Table......58
Table 6-1......58
Table 6-2......59
Table 6-3......60
References......61

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