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研究生:吳崇吉
研究生(外文):Chong-Ji Wu
論文名稱:鏈堆積導致的彎曲模量變化對Alamethicin離子通道活性的影響
論文名稱(外文):The Effects of Chain Packing-induced Variation in Bending Modulus on the Ion Channel Activity of Alamethicin
指導教授:陳儀帆
指導教授(外文):Yi-Fan Chen
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程學系
學門:工程學門
學類:化學工程學類
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:101
中文關鍵詞:離子通道彎曲模量鏈堆積Alamethicin
外文關鍵詞:Ion Channel ActivityBending ModulusChain PackingAlamethicin
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自發曲率(spontaneous curvature)和彎曲模量(bending modulus)是生物膜的兩個主要彈性特性。兩種彈性均受到鏈堆積的影響,其中,鏈堆積會影響彎曲模量。Keller和其團隊提及,膜的自發曲率決定了由肽(阿樂美黴素)形成的離子通道的活性。然而,由鏈堆積所引起膜的彎曲模量是否以及如何影響離子通道的活性仍然未知。為了回答這個問題,我們採用了由單不飽和脂質(DOPC和DOPS)以及由多不飽和脂質(di (18:2) PC 和DOPS)構成的脂質囊泡,因為兩種囊包組成的彎曲模量不同但自發曲率不同,並且觀察當囊泡組成發生變化時,Alamethicin離子通道的活性如何被調節。我們採用動態光散射和定向圓二色性來確定囊泡的大小和阿樂美黴素在脂質膜中的方向,同時通過將鈣敏感染劑fura-2包裹在囊泡中,通過螢光光譜法測量離子通道的活性,以檢測通過通道的鈣通量。我們發現受膜影響的彎曲模量對阿樂美黴素離子通道的活性是有影響的,這可能歸因於膜中脂質分子的堆積。
Spontaneous curvature and bending modulus are the two major elastic properties of biological membranes. Both of elastic properties are influenced by chain packing. Among them, the chain packing would influence bending modulus. Keller and coworkers reported that the spontaneous curvature of a membrane dictated the activity of the ion channels formed by the peptide, alamethicin. However, whether and how the chain packing-induced variation in bending modulus of a membrane affects the activity of ion channels remains unknown. To answer this question, we employed the lipid vesicles made of the monounsaturated lipids, DOPC and DOPS, as well as those made of polyunsaturated lipids, di (18:2) PC and DOPS, and observed how the activity of the alamethicin ion channels is modulated when the vesicle composition changes, since the vesicles of the two compositions differ in bending modulus but not in spontaneous curvature. We employed dynamic light scattering and orientation circular dichroism to determine the vesicle size and the orientation of alamethicin within the membranes, while the activity of the ion channels was measured with fluorescence spectroscopy via enveloping the calcium-sensitive dye, fura-2, into the vesicles to detect the calcium flux passing the channels. We found that the bending modulus of a membrane affected was influential to the activity of the alamethicin ion channels, which is possibly ascribable to the packing of the lipid molecules in a membrane.
摘要 I
ABSTRACT II
致謝 III
TABLE OF CONTENTS IV
LIST OF FITURES VII
LIST OF TABLES IX
CHAPTER 1 INTRODUCTION 1
1-1 Membrane system 2
1-1-1 Cell membrane 2
1-1-2 Phospholipids 4
1-1-3 Biomimetic membrane 6
1-2 Elastic properties 7
1-2-1 Spontaneous curvature 10
1-2-2 Bending modulus 12
1-3 Antimicrobial peptides (AMPs) 14
1-3-1 Alamethicin 14
1-4 Mechanism of peptide and membrane 16
1-4-1 peptide adsorption and insertion 18
1-4-2 Ion channel 21
1-5 Motivation 21
CHAPTER 2 MATERIALS AND INSTRUMENTS 23
2-1 Materials 23
2-1-1 Phospholipid 24
2-1-2 Peptide 27
2-1-3 Fluorescence dye 30
2-1-4 General materials 31
2-2 Sample preparation 34
2-2-1 Buffer preparation 34
2-2-2 Peptide solution preparation 34
2-2-3 Unilamellar vesicle 35
2-2-4 Vesicle enveloping fluorescence dye and leakage 36
2-2-5 Supported lipid bilayer 37
2-3 Experiment instruments and methods 39
2-3-1 Small angle X-ray scattering (SAXS) 39
2-3-2 Orientation circular dichroism (OCD) 41
2-3-3 Gel filtration chromatography 45
2-3-4 Absorption fluorometer 47
2-3-5 Dynamic light scattering (DLS) 50
2-4 Data analysis 51
2-4-1 SAXS data analysis 51
2-4-2 Fluorescence dye leakage analysis 54
CHAPTER 3 RESULT 56
3-1 Particle size of vesicles 56
3-2 Orientation of peptide on different bending modulus of phospholipid 58
3-3 Influence of different bending modulus on ion channels activity. 61
3-3-1 The range of volume of elution contained Fura-2 and vesicle 61
3-3-2 The measurable method of fluorescence assay 65
3-3-3 The activity of ion channel in DOPC/DOPS and di (18:2) PC/DOPS 67
3-4 The relation between bending modulus and membrane thickness after peptide binding 69
CHAPTER 4 DISCUSSION 75
CHAPTER 5 CONCLUSION 77
REFERENCE 78
APPENDIX 83
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