博碩士論文 106324018 詳細資訊




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姓名 魏振豪(Chen-Hao Wei)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 抗肌萎縮蛋白的膜結合錨如何影響其與脂質膜的相互作用
(INVESTIGATION ON HOW THE MEMBRANE-BINDING ANCHOR OF DYSTROPHIN AFFECTS ITS INTERACTIONS WITH LIPID MEMBRANES)
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摘要(中) 抗肌萎縮蛋白(dystrophin)是連接F-肌動蛋白(F-actin)與肌肉細胞膜的一種蛋白質,主要功能在於穩定肌肉細胞膜使其免於肌肉拉伸所造成的損害。人體缺乏抗肌萎縮蛋白時會導致杜興氏肌肉營養不良症(Duchenne muscular dystrophy),其病症為肌肉收縮時的肌肉無力和肌肉退化,肇因可能來自於抗肌萎縮蛋白與膜之間交互作用上的異常。抗肌萎縮蛋白由四個結構域(domain)所組成:與肌動蛋白連結結構域(actin binding amino-terminal domain)、包含24個血影蛋白樣重複區段(spectrin-like repeats)的棒狀結構域(rod domain)、與膜連結並富含半胱氨酸(cysteine)的錨定結構域(cysteine-rich membrane-binding anchor domain)以及C末端結構域(C-terminal domain)。先前的研究指出,特定之抗肌萎縮蛋白之棒狀結構域會與脂質膜產生相互作用,因此具有生物功能上的重要性。然而,這類研究皆是使用不含錨定域的抗肌萎縮蛋白異構體來進行。為了探討錨定域對抗肌萎縮蛋白與膜間之交互作用的影響、並更真實地模擬生物系統,本研究使用棕櫚酸作為抗肌萎縮蛋白異構體之錨定域。此錨定域成功並有效地增強了抗肌萎縮蛋白異構體與膜之間的結合作用,但作用過程中,脂質膜的結構並沒有明顯的改變。錨定域對於抗肌萎縮蛋白異構體與脂質膜之間的作用能力已在此研究中被證實,但背後的機制仍未被完全了解。
摘要(英) Dystrophin is a protein which connects the F-actin (a composing protein of microfilaments) and muscle cell membrane through the protein complex. Lack of dystrophin will lead to Duchenne muscular dystrophy (DMD), which features muscle weakness and muscle degeneration and may involve the abnormality of the dystrophin-membrane interactions. The dystrophin is composed of four domains: an actin binding domain, a rod domain comprising 24 spectrin-like repeats, a cysteine-rich domain and a C-terminal domain. In recent decades, some specific repeats of rod domain were demonstrated to interact with lipid membranes. But those experiments were done in a compromising condition which using dystrophin isoform without the anchor part. To explore how the presence of anchor domain affects the dystrophin-membrane interactions and to more resemble a real living system, we introduce a palmitic acid onto C-terminal of dystrophin isoform to serve as an anchor. The anchor make a pronounced effect in our series of experiments, which intensely reinforces the binding ability of dystrophin isoform to the lipid membrane. But the conformation of membrane wasn’t change obviously during the interaction. Although the excellent effect of palmitic acid anchor had been proved in this study. The mechanism working inside is still a puzzle waited to be unrevealed.
關鍵字(中) ★ 抗肌萎縮蛋白
★ 脂質膜
★ 錨定域
關鍵字(英) ★ Dystrophin
★ Lipid membrane
★ Anchor domain
論文目次 摘要 VI
Abstracts VII
致謝 VIII
TABLE OF CONTENTS IX
LIST OF FIGURES XI
LIST OF TABLES XIII
LIST OF EQATIONS XIV
CHAPTER 1 INTRODUCTION 1
1-1 Dystrophin 1
1-2 Duchenne muscular dystrophy (DMD) 3
1-3 Gene delivery therapy 6
1-4 Cell membrane and biomimetic system 8
1-4-1 Lipid molecules 9
1-4-2 Human muscle cell membrane 11
1-4-3 Artificial membrane 12
1-5 Motivation 14
CHAPTER 2 MATERIALS AND INSTRUMENTS 17
2-1 Materials 17
2-1-1 Phospholipids 19
2-1-2 Peptides 21
2-1-3 General Chemicals 27
2-2 Sample preparation 29
2-2-1 Buffer lyophilization 29
2-2-2 Vesicle preparation 30
2-2-3 Peptide solution preparation 31
2-3 Instruments 32
2-3-1 Circular dichroism 32
2-3-2 Langmuir trough 35
2-3-3 Dynamics light scattering 41
2-3-4 Isothermal titration calorimetry 42
2-3-5 Ultraviolet-visible spectroscopy 45
2-3-6 Small angle X-ray scattering, SAXS 47
2-3-7 Small angle neutron scattering, SANS 52
2-4 Data analysis 57
2-4-1 SAXS data analysis 57
2-4-2 SANS data analysis 61
CHAPTER 3 RESULT 63
3-1 Determination of vesicle particle size 63
3-2 Characterization of dystrophin isoform 65
3-3 Secondary structural change during interaction 67
3-4 Oxidation of lipid 69
3-5 Lipid isotherm 74
3-6 Interaction between lipid monolayer and dystrophin isoform 78
3-7 Determination of peptide concentration 87
3-8 Thermodynamic change during interaction 88
3-9 Morphology change of vesicle after interaction with dystrophin isoforms 94
CHAPTER 4 DISCUSSION 104
CHAPTER 5 CONCLUSION 108
REFERENCE 110
APPENDIX 114
The asymmetric membrane model (Matlab code) 114
SAXS Average fitting result of symmetric membrane model (GAP) 119
SAXS Average fitting result of asymmetric membrane model 121
SANS Average fitting result 122
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指導教授 陳儀帆(Yi-Fan Chen) 審核日期 2019-8-24
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