添加具有抗菌潛力的胜肽對磷脂質自組裝結構與彈性性質的影響

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張雯芳(Wen-Fang Chang) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

添加具有抗菌潛力的胜肽對磷脂質自組裝結構與彈性性質的影響
(INFLUENCES OF ANTIMICROBIAL-POTENT PEPTIDES ON THE STRUCTURES AND ELASTIC PROPERTIES OF THE PHOSPHOLIPID SELF-ASSEMBLIES)

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摘要(中)

抗菌肽（Antimicrobial peptides）為生物體先天性免疫反應的一環，可對抗病原體及癌細胞，且其透過擾動細胞膜的方式作用，較不易產生抗藥性，因此被認為是具有潛力的新式抗生藥物。為更瞭解抗菌肽與細胞膜的作用機制，本實驗探討了抗菌肽中富含最多的胺基酸-精胺酸（arginine, R）對磷脂質（phospholipids）自組裝結構的物理及結構性質的影響，並從中進一步討論peptide對磷脂質膜的擾動機制。本實驗使用了4種poly-Arginine peptide ─ R6、R9、R6C及R9C，以探討不同poly-Arginine peptide的長度及其尾端接上另一胺基酸 ─ Cysteine對磷脂質自組裝結構的影響。本研究利用2種不同的磷脂質自組裝結構（self-assembled structure）進行實驗：我們首先選用了易形成inverted hexagonal phase的1,2-dioleoyl-sn-glycero-3-phosphoethanolamine（DOPE）進行實驗，並利用Small-Angle X-Ray Scattering（SAXS）探討添加peptide對DOPE彈性性質（elastic properties）的作用。另一方面，為了瞭解peptide如何影響細胞膜(cell membrane)，我們亦使用24 mol% DOPE/72 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine（DOPC）/4 mol% 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate（DOPA）製備模擬細胞膜的微脂體（liposome），以探討peptide如何改變liposome的膜厚及結構穩定性。實驗結果發現，R6、R9、R6C的添加會使得DOPE自組裝結構的一項彈性性質 ─ spontaneous curvature變小，而R9C則會使其變大；另一方面， bending modulus這項彈性性質則會因R6及R6C的添加而變小（意即膜變得較為柔軟），因R9的添加而變大（意即膜變得較為堅硬）。我們根據實驗結果了解：(1)親水性的poly-Arginine peptide確實可與不帶淨電荷的磷脂質 ─ DOPE相互作用；(2)不同的poly-Arginine peptide對DOPE自組裝結構之spontaneous curvature及bending modulus的影響可能與其嵌入該自組裝結構的深度有關；(3)不同長度之poly-Arginine peptide可能以不同的機制對liposome的結構進行擾動。

摘要(英)

Antimicrobial peptides are important players in the immune systems and exert their influences mainly through their interactions with biomembranes. Due to their high efficiency in killing diverse invading molecules/organisms and low vulnerability to the drug resistance, antimicrobial peptides have drawn considerable attention and been regarded as promising candidates for the therapeutics of next generation. In order to improve our understanding toward the mechanisms underlying the interactions between antimicrobial peptides and biomembranes, this thesis work studied how the amino acid enriched in antimicrobial peptides, arginine, affects the physical and structural properties of phospholipid self-assemblies. Four types of poly-arginine peptides, R6, R9, R6C and R9C, were chosen for this study to scrutinize how the peptide chain length and the addition of the amino acid, cysteine, to the C-termini of the peptides modulate the properties of a phospholipid self-assembly. Two self-assembled structures were prepared for our research: First, the inverted hexagonal phase, prepared with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was required for the small-angle X-ray scattering (SAXS) measurements of the elastic properties; second, 24 mol% DOPE/72 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/4 mol% 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate (DOPA) were used to prepare the cell membrane-mimicking liposomes (unilamellar vesicle), with their membrane thickness and structural stability investigated in order to extract the information on how the peptides interact with cell membranes. Our experimental results indicate that the presence of R6, R9 and R6C decreased the spontaneous curvature (an elastic property, which quantified the tendency of forming nonlamellar phases of a lipid) of DOPE, whereas the bending modulus (another elastic property, which described how hard the membrane is against bending) of DOPE was increased (i.e., the phospholipid self-assembly was stiffened) upon the addition of R9 and decreased (i.e., the phospholipid self-assembly was softened) upon the addition of R6 and R6C. Based on our results, we conclude: (1) Poly-arginine peptides have considerable interactions with DOPE even though the peptides carry no hydrophobic residue and the lipid self-assembly is electrostatically neutral (both of which point to the lack of attractive forces between the peptides and the self-assemblies); (2) how the different poly-arginine peptides modulate the spontaneous curvature and the bending modulus is speculated to depend on the insertion depths into the lipid self-assemblies of the peptides; (3) different underlying mechanisms may be employed by the poly-arginine peptides of different chain lengths to disrupt the structures of liposomes.

關鍵字(中)

★ 抗菌肽
★ 磷脂質
★ 自組裝結構
★ 彈性性質
★ 小角度X光散射
★ 微脂體

關鍵字(英)

★ Antimicrobial peptides
★ phospholipids
★ Self-Assemblies
★ Elastic Properties
★ Small-Angle X-Ray Scattering（SAXS）
★ liposome

論文目次

摘要...................................................I
Abstract..............................................III
致謝...................................................V
目錄..................................................VII
圖目錄..................................................X
表目錄...............................................XVII
第一章緒論...........................................1
1.1 細胞膜（Biomembrane）................................2
1.2 磷脂質（phospholipids）..............................5
1.3 抗菌肽（Antimicrobial peptides）....................11
1.4 抗菌胜肽與細胞膜作用機制..............................15
1.5 磷脂質自組裝結構（Self-assembled structure）.........24
1.6 彈性性質（Elastic properties）......................29
1.6.1 自發曲率（Spontaneous curvature, C0）.............30
1.6.2 彎曲係數（Bending modulus, Kcp）..................32
1.7 研究動機與目的......................................34
第二章實驗材料及方法....................................35
2.1 實驗材料............................................35
2.1.1 磷脂質（Phospholipid）............................35
2.1.2 Peptide..........................................38
2.1.3 實驗藥品..........................................41
2.2 實驗器材............................................43
2.3 實驗儀器............................................45
2.4 實驗步驟............................................46
2.4.1 Dispersion (Reverse hexagonal phase, HII)........46
2.4.2 Liposome method..................................50
2.5 儀器分析............................................55
2.5.1 Small-Angle X-Ray Scattering（SAXS）.............55
2.5.2 熱示差掃瞄卡量計（Differential scanning calorimetry, DSC）..................................................57
2.5.3 圓二色光谱（Circular dichroism spectroscopy, CD）.59
2.5.4 螢光（Fluorescence）..............................61
2.6 X繞射實驗（X-ray diffraction）......................62
2.7 螢光洩漏實驗（Peptide-induced membrane leakage）.....64
2.8 滲透壓測量實驗（Peptide binding measurements）.......65
2.9 數據處理與分析......................................66
2.9.1 重建HII phase電子雲密度（The electron density profile reconstruction of reverse hexagonal phase, HII ）......66
2.9.2 量化自發曲率（Quantified of spontaneous curvature, C0）...................................................71
2.9.3 Liposome結構參數..................................73
第三章結果.............................................78
3.1 Peptide對DOPE spontaneous curvature的影響...........80
3.1.1 不同poly-Arg peptide對DOPE spontaneous curvature的影響.....................................................80
3.1.2 Indolicidin及SAP10-C對DOPE膜spontaneous curvature的影響...................................................84
3.2 Peptide對DOPE膜bending modulus（Kcp）的影響.........88
3.3 Peptide結構的變化...................................92
3.3.1 R6、R9及R6C的結構.................................92
3.3.2 Liposome的存在對peptide結構變化....................94
3.4 Peptide對liposome結構及膜厚（thickness）的影響.......96
3.5 Peptide對liposome結構穩定性的影響...................100
3.6 Peptide對DOPE膜thermotropic behavior的影響.........103
3.6.1 DOPE膜packing frustration影響...................103
3.6.2 Peptide對沒有packing frustration的DOPE膜的影響....105
3.6.3 Peptide對有packing frustration的DOPE膜之影響.....107
第四章討論............................................109
4.1 Peptide與membrane的交互作用力......................110
4.2 添加peptide對DOPE膜的彈性能探討（Elastic energy of the DOPE membrane）.......................................116
4.3 探討peptide與membrane作用機制......................118
第五章結論............................................121
參考文獻 ..............................................124

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指導教授

陳儀帆(Yi-Fan Chen)

審核日期

2015-7-29

推文