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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 | Si-Han Chen | en |
dc.contributor.author | 陳熙函 | zh_TW |
dc.date.accessioned | 2021-06-08T06:25:10Z | - |
dc.date.copyright | 2006-07-31 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25693 | - |
dc.description.abstract | Lon蛋白水解酵素廣泛存於各物種中,從古生菌及原核生物到人類粒線體均先後被鑑定出Lon的存在,且其序列與功能具高度保留性,目前研究顯示Lon對於細胞恆定維持及演化上可能極為重要。Lon目前已被發現具有多種功能,包括proteolytic, ATPase, chaperone及DNA-binding等活性。 然而對於Lon本身結合DNA的特性與意義,包括是否具有專一辨認的序列,至今仍尚未被釐清。
本篇論文旨在以熱力學的角度,探究人類Lon (huLon)與DNA結合的物化特性。文中主要探討反應焓變化(ΔH) 、熵變化(ΔS) 、自由能變化(ΔG)及結合常數(K)對溫度的依賴性,以TH與TS作為標的溫度(TH定義為ΔH為零時的溫度,而TS則定義為ΔS為零時的溫度),分析高低溫時反應驅使力(thermodynamic driving forces)的差異,配合反應熱容變化(heat capacity change)平行討論,進而剖析Lon對不同寡核酸鏈(DNA oligonucleotides)間之結合強度與專一性的差異。其中寡核酸鏈自身的二級結構 — G-quartet於huLon對DNA結合特性之關連性為何,亦是本文探討的重點。 構成本篇論文的核心實驗是以恆溫滴定熱卡計(Isothermal Titration Calorimeter, ITC)量測Lon與寡核酸DNA鏈結合之熱變化(單位為μwatt),而該值對時間積分後除以滴定液的莫耳數,得每個滴定反應之熱變化值(cal/mol),便得到等溫結合反應曲線(binding isotherm),該曲線以非線性迴歸方法求得可信賴之焓變化與平衡結合常數,並以所得常數進而推算熵變化、自由能變化以及反應熱容變化。以這些熱力學常數為基礎,我們發現huLon對於先前研究指出huLon可能的結合目標,24-mer LSPas (5’-AATAATGTGTTAGTTGGGGGGTGA-3’),其結合反應主要由焓變化所驅使,且隨著高溫影響更鉅,然而其自由能變化並不隨溫度而異,由反應之各能量項對溫度作圖,揭露該結合反應具相當強的焓熵互補效應,並伴隨呈極負值的反應熱容變化(-607.82±31.39 cal?mol-1?K-1),這些均是專一性結合的特徵。另一方面,huLon與8-mer TG6T (5’-TGGGGGGT-3’)結合反應雖然具有與24-mer LSPas近乎相同的K與ΔG,但其焓、熵對溫度的依賴性卻與後者迥異,huLon結合至8-mer TG6T主要由熵變化所驅使,其焓變化負值程度隨溫度升高而變小,但改變幅度並不大,因此反應熱容變化相較之下也很小 (67.29±4.52 cal?mol-1?K-1),屬於非專一性結合。 我們進一步以差異性掃描式熱卡計 (Differential Scanning Calorimeter, DSC)檢驗huLon、DNA、與各huLon-DNA複合物的熱容量,顯示huLon惟有與24-mer LSPas形成複合物時,才具有額外的熱容量(excess heat capacity),這也驗證先前以ITC得到的結果。本篇論文研究顯示,G-quartet的高度負電密度結構主要以靜電交互作用力(electrostatic interaction)與huLon,以穩定huLon-DNA複合物,然而這種結合模式易受環境(如溫度與離子強度)所影響,無法構成專一性結合。然而從粒線體DNA鹼基組成偏差現象分析,G-quartet可能扮演引導huLon至專一結合區段之重要角色。 在本研究中鑑定之特殊結合區段,24-mer LSPas在粒線體DNA(mtDNA)中座落在displacement loop (D-loop)區域,D-loop是長達0.5~1 Kb的noncoding region,於mtDNA複製的調節極為重要,目前研究已知mtDNA的複製會因應微環境中代謝的改變,異動其複製起始點(replicational origins),而huLon是否藉由結合至LSPas這段區域以調節mtDNA複製,絕對值得我們高度關切。 | zh_TW |
dc.description.abstract | Lon is a class of ATP-dependent protease, which is committed to several crucial functions including proteolytic, ATPase, chaperone, and DNA-binding activities. Previously, human Lon protease (huLon) has been reported that it binds to an element of antisense light strand promoter (5’-AATAATGTGTTAGTTGGGGGGTGA-3’, designated as 24-mer LSPas) within the displacement loop (D-loop) region of mitochondrial genome. In this study, the potential specific binding site for huLon containing six consecutive guanine bases, 24-mer LSPas, was found to form G-quartet in various ionic conditions with electrophoretic and circular dichroism (CD) spectroscopic analysis. We have investigated the thermodynamics of the binding reactions of huLon to 24-mer LSPas and the G-quartet-forming unit (5’-TGGGGGGT-3’, or simply called 8-mer TG6T) respectively by using isothermal titration calorimetry (ITC). The results revealed that the interactions between huLon and 24-mer LSPas are primarily driven by enthalpy change, which exhibits strong dependence on temperature, rendering a large negative heat capacity, -607.82±31.39 cal?mol-1?K-1. Though, the binding of 8-mer TG6T to huLon is quite comparable in binding constant and free energy change with that of 24-mer LSPas. In contrast to what was observed in the binding reaction of 24-mer LSPas, the binding of huLon to 8-mer TG6T is mainly entropically driven, and associated with a small change in the heat capacity of 67.29±4.52 cal?mol-1?K-1, which reflects a lack of strong enthalpy-entropy compensation.
The thermal property of each binding component as well as complex was further examined with differential scanning calorimetry (DSC). We observed a DNA-dependence of excess molar heat capacity of huLon when it is in the presence of 24-mer LSPas. The results are consistent with what were observed in the ITC experiments. We concluded that the polyanionic G-quadruplex with high charge density interacts with huLon with appreciable affinity but in a nonspecific manner. Our thermodynamic analysis has revealed that huLon specifically binds 24-mer LSPas, and accompanies structural rearrangement, which is also indicated in the CD experiments. By evaluating the entropic cost, we found that about 32 residues of huLon are coupled with folding to the site-specific binding. The energetic interpretations may shed new light on the DNA-binding mechanism of human Lon protease. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T06:25:10Z (GMT). No. of bitstreams: 1 ntu-95-R93b46005-1.pdf: 162917708 bytes, checksum: 8141cd84179314ad4d802be3a037f984 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | Table of Contents
中文摘要 i Abstract iii Abbreviation Table v Chapter 1 Introduction 1.1 Background Overview 1 1.2 Motivation and Aim of Research 4 Chapter 2 Materials and Experimental Procedures 2.1 Expression and Purification of Human Lon Protease 11 2.2 Protease Assay 13 2.3 Oligonucleotides 14 2.4 Native and Denaturing Polyacrylamide Gel Electrophoresis 14 2.5 Circular Dichroism (CD) Spectroscopy 15 2.5.1 The Instrumentation 2.5.2 Experimental Procedures 2.6 Isothermal Titration Calorimetry (ITC) 18 2.6.1 The Instrumentation 2.6.2 Experimental Procedures 2.7 Differential Scanning Calorimetry (DSC) 20 2.7.1 The Instrumentation 2.7.2 Experimental Procedures 2.8 Electrophoresis Mobility Shift Assay (EMSA) 21 Chapter 3 Results and Discussions 3.1 Purification of Recombinant Human Lon and Analysis of Its Protease Activity 25 3.2 A Low Mobility Form of LSPas Is Observed in The Electrophoretic Analysis 25 3.3 Formation of G-quadruplex in LSPas Is Characterized with Circular Dichroism Spectroscopy 28 3.4 Thermodynamic Characterization of The Binding of Human Lon to 24-mer LSPas and 8-mer TG6T 30 3.4.1 Thermal Properties of the Binding Components 3.4.2 Sequence-specific Binding of Human Lon Protease to 24-mer LSPas Is Characterized by Both Large Negative Heat Capacity Change and Reaction enthalpy 3.4.3 Human Lon Protease Binds to 8-mer TG6T in A Nonspecific Manner 3.4.4 DNA-dependence of Excess Molar Heat Capacity of Human Lon Is Also Observed in The Differential Scanning Calorimetric Analysis 3.4.5 Statistical Thermodynamics for The Coupled Folding of Human Lon to Site-specific Binding 3.5 Electrophoresis Mobility Shift Assay Reveals that Human Lon Preferentially Binds to G-quartet 40 3.5.1 The G-rich DNA Sequences Present in Human Mitochondrial DNA Exhibit High-ordered Structures in Native PAGE 3.5.2 Human Lon Preferentially Binds to G-quartet 3.6 G-quartet May Play A Role in Conducting Human Lon to The Specific Binding Site(s) 43 Chapter 4 Conclusions 70 Chapter 5 References 71 Appendage Poster (The Best Poster Award in the 11th Symposium on Recent Advances in Biophysics) ----------------------- List of Tables and Figures Chapter 1 Introduction Figure 1-1 Sequence alignment of Lon proteases 8 Figure 1-2 Domain structures of Lon protease 9 Figure 1-3 Schematic diagram of the functions of Lon 9 Figure 1-4 Mitochondrial defects caused by Lon downregulation 10 Figure 1-5 Replication of mitochondrial DNA 10 Chapter 2 Materials and Experimental Procedures Framework 23 Framework (cont’d) 24 Chapter 3 Results and Discussions Figure 3-1 SDS-PAGE of Ni2+-column-purified human Lon 47 Figure 3-2 ATP-dependent protease activity of human Lon 47 Table 3-1 The DNA oligonucleotides 48 Figure 3-3 Schematic representation of mitochondrial DNA 48 Figure 3-4 The D-loop region of mitochondrial DNA 49 Figure 3-5 G-quartet structure 50 Figure 3-6 Electrophoresis of the oligonucleotides 51 Figure 3-7 Electrophoresis of the oligonucleotides 52 Figure 3-8 The CD spectra of 24-mer LSPas in native and denaturing buffers 53 Figure 3-9 The CD spectra of 8-mer TG6T in native and denaturing buffers 54 Figure 3-10 The CD spectra of human Lon 55 Figure 3-11 The thermal profiling of human Lon in CD spectroscopy 56 Table 3-2 Thermodynamic parameters for the binding of human Lon to 24-mer LSPas and 8-mer G-quartet-forming unit respectively 57 Figure 3-12 Schematic representation of the thermodynamic parameters (ΔS, ΔH, and ΔCp), which can be partitioned to several contributing factors for DNA-protein complexation 57 Figure 3-13 Isothermal titration calorimetric analysis for the binding of huLon to 24-mer LSPas 58 Figure 3-14 Temperature profile of the energy plot for the binding of huLon to 24-mer LSPas 59 Figure 3-15 Isothermal titration calorimetric analysis for the binding of huLon to 8-mer TG6T 60 Figure 3-16 Temperature profile of the energy plot for the binding of huLon to 8-mer TG6T 61 Figure 3-17 The CD spectra of human Lon in presence of DNA 62 Figure 3-18 The summary of the binding thermodynamic patterns 63 Figure 3-19 The heat capacities of human Lon in the presence and absence of DNA 64 Figure 3-20 The heat capacities of human Lon in the presence of various G-rich DNA oligonulceotides 65 Table 3-3 The G-rich DNA sequences present in human mtDNA 66 Figure 3-21 Nondenaturing PAGE of the G-rich DNA sequences 66 Figure 3-22 Electrophoresis mobility shift assay for detecting the binding of human Lon to each DNA sequence Figure 3-23 The DNA competitors visualized through UV shadowing 68 Figure 3-24 Model for the nonspecific and specific binding of human Lon to DNA 69 | |
dc.language.iso | en | |
dc.title | 人類Lon蛋白水解酵素對DNA結合特性之熱力學研究 | zh_TW |
dc.title | Thermodynamic Characterization of DNA-binding Activity of Human Lon Protease | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張文章(Wen-Chang Chang),甘魯生(Lou-Sing Kan),張家靖(Chia-Ching-Chang) | |
dc.subject.keyword | 粒線體DNA,蛋白水解酵素,生物熱力學,DNA四重螺旋,端粒,端粒結合蛋白,DNA結合蛋白,熱容,焓,亂度,熵,恆溫滴定熱卡計,差異性掃描熱卡計,圓二色光譜, | zh_TW |
dc.subject.keyword | Lon protease,DNA-binding protein,thermodynamic driving force,G-quartet,G-quadruplex,G-quartet-binding protein,mitochondrial DNA,D-loop,hydrophobic interactions,heat capacity,enthalpy,entropy,isothermal titration calorimetry,differential scanning calorimetry,circular dichroism, | en |
dc.relation.page | 75 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2006-07-29 | |
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 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-95-1.pdf 目前未授權公開取用 | 159.1 MB | Adobe PDF |
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