臺灣博碩士論文加值系統

Lon蛋白為一個多功能的單一聚合型酵素，且高度保留存在於各種生物體中。先前的研究指出Lon可以維持生物體中蛋白質完整的功能與結構，或是適時降解特定或非特定目標蛋白，進而參予調控體內多種新陳代謝活動。本論文以台灣本土所分離出的嗜熱短桿菌(Brevibacillus thermoruber WR-249)之Lon蛋白(Bt-Lon)為研究對象，探討其與DNA結合之間的關係，並同時與革蘭氏陽性標準菌株-枯草桿菌(Bacillus subtilis)及革蘭氏陰性標準菌株-大腸桿菌(Escherichia coli)之Lon蛋白進行比較，利用電泳凝膠遲滯法(GMSA)分析，得知原核細菌的Lon蛋白主要皆是利用其中的α-domain為DNA結合區域。利用DNase I配合基質輔助電射游離脫附飛行時間質譜(MALDI-TOF MS)分析，我們找到一段與Bt-Lon α-domain結合的DNA片段(5’-CTGTTAGCGGGC-3’)，我們將之命名為ms1。再利用表面電漿共振(SPR)及恆溫滴定熱分析(ITC)，確認ms1與Bt-Lon α-domain之間具有高專一性結合能力。參考Bt-Lon α domain結構的表面正電荷分佈，我們針對其中部份帶正電荷之胺基酸殘基進行定點突變，再進行與ms1 DNA之結合分析，結果顯示當Arginine 518殘基突變為丙胺酸後，結合力會明顯下降26倍，顯示在Bt-Lon α-domain與ms1 DNA之間的結合上，Arginine 518扮演重要的角色。

The multi-functional, homo-oligomeric, ATP-dependent Lon protease is highly conserved in prokaryotes and eukaryotic organelles. Previous studies have shown that Lon activity is essential for protein quality control and regulation of metabolic processes. Here we examined the DNA-binding activity of the Lon protease α-domains from Brevibacillus thermoruber, Bacillus subtilis, and Escherichia coli. Gel mobility shift assays indicated that the α-domain from Br. thermoruber has the highest DNA affinity. MALDI-TOF mass spectrometry showed that this α-domain binds to the nucleotide sequence 5’-CTGTTAGCGGGC-3’ (ms1). Surface plasmon resonance and isothermal titration calorimetry showed that a double-stranded DNA fragment of this sequence binds to the α-domain; double-stranded DNA fragments with 0 and 50% identity to the binding sequence had lower affinities for the α-domain. Five mutants of the α-domain from Br. thermoruber carrying single mutations (R537A, R546A, R553A, K580A and R584A) were constructed and showed only 1.2–2.0-fold lower DNA binding affinity; one mutant, R518A, displayed 26-fold lower affinity. The Bt-Lon R518A mutant also has lower affinity to DNA than wild type. These results revealed that Arg 518 of the Bt-Lon from Br. thermoruber plays a critical role in the DNA-binding activity.

口試委員會審定書 i
中文摘要 ii
Abstract iii
1. Introduction - 1 -
2. Materials and Methods - 6 -
2-1. Subcloning of the α-domains - 6 -
2-2. Sequence alignment - 6 -
2-3. Expression and purification of Bt-Lon truncated proteins and α-domains - 6 -
2-4. Gel mobility shift assay - 7 -
2-5. Identification of the Bt-Lon α-domain DNA-binding sequence - 8 -
2-6. Homology modeling - 9 -
2-7. Site-directed mutagenesis - 9 -
2-8. Circular dichroism - 10 -
2-9. Surface hydrophobicity - 11 -
2-10. Native-PAGE of hairpin DNA - 11 -
2-11. Surface plasmon resonance (SPR) - 11 -
2-12. Isothermal titration calorimetry - 13 -
2-13. Protease assay - 13 -
2-14. Peptidase assay - 14 -
2-15. ATPase assay - 14 -
2-16. Docking model of protein and DNA complex - 15 -
3. Results - 17 -
3-1. Lon α-domains from Br. thermoruber, B. subtilis, and E. coli - 17 -
3-2. Structure of the α-domains - 18 -
3-3. Binding of the α-domain to DNA - 18 -
3-4. The sequence of the preferred DNA binding site of the Bt-Lon α-domain - 19 -
3-5. Kinetics of the interaction between the wild-type α-domain and DNA - 20 -
3-6. Thermodynamics of the interaction between the α-domain and DNA - 21 -
3-7. Interaction between DNA and Bt-Lon α-domain mutants - 21 -
3-8. DNA sequence specificity of Bt-Lon α-domain binding - 23 -
3-9. Interaction between DNA and Bt-Lon wild-type and R518A mutant - 23 -
3-10. Influence of plasmid or ms1 DNA on enzymatic activities of Bt-Lon - 24 -
3-11. The structure model of Bt-Lon α-domain/ds-ms1 complex - 25 -
4. Discussion - 26 -
Figure - 36 -
Figure 1. The amino acid sequence alignment of α-domains - 36 -
Figure 2. SDS-PAGE of purified recombinant α-domains - 37 -
Figure 3. Structure of α-domains - 38 -
Figure 4. Far-UV CD spectra - 39 -
Figure 5. Thermal denaturation - 40 -
Figure 6. Near-UV spectra - 41 -
Figure 7. Hydrophobicity measurement - 42 -
Figure 8. Concentration dependent EMSA assay - 43 -
Figure 9. GMSA assay of different α-domains - 44 -
Figure 10. MALDI-TOF mass spectroscopy - 45 -
Figure 11. Analysis of hairpin-duplex formation by Native PAGE - 46 -
Figure 12. SPR sensorgrams of different α-domains - 47 -
Figure 13. ITC result of the Bs-Lon α-domain - 48 -
Figure 14. ITC result of the Bt-Lon α-domain - 49 -
Figure 15. ITC result of the Ec-Lon α-domain - 50 -
Figure 16. Site directed mutagenesis - 51 -
Figure 17. SDS-PAGE of Bt-Lon α-domain mutants - 52 -
Figure 18. Far-UV CD spectra of 6 different Bt-Lon α-domain mutants - 53 -
Figure 19. SPR sensorgrams of Bt-Lon α-domain mutants - 54 -
Figure 20. SPR sensorgrams of Bt-Lon α-domain to different hairpin DNA - 55 -
Figure 21. GMSA assay of Bt-Lon wild type and mutant - 56 -
Figure 22. SPR sensorgrams of the binding of Bt-Lon - 57 -
Figure 23. Effect of plasmid DNA on enzymatic activities - 58 -
Figure 24. Effect of ds-ms1 DNA on enzymatic activities - 59 -
Figure 25. Structural model of Bt-Lon α-domain / ds-ms1 DNA - 60 -
Table - 61 -
Table 1. Oligonucleotides used in this study - 61 -
Table 2. The ratios of the secondary structures in different α-domains - 62 -
Table 3. Kinetic constants of the interaction - 63 -
References - 64 -
Appendix - 72 -
Paper List - 72 -
In Preparation List - 72 -
Poster List - 73 -
Paper - 74 -
Poster - 107 -

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