使用指向性圓二色光譜和多片層X光繞射研究Magainin 2與混合脂質雙層膜的交互作用

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江佳蓉(Chia-Jung Chiang) 查詢紙本館藏

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生物物理研究所

論文名稱

使用指向性圓二色光譜和多片層X光繞射研究Magainin 2與混合脂質雙層膜的交互作用
(Studying on interaction between Magainin 2 and mixed lipids bilayer by Oriented Circular Dichroism (OCD) and Lamellar X-ray Diffraction (LXD))

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摘要(中)

抗菌胜肽是生物免疫系統中殺死入侵微生物的利器，其殺菌方式並非與生物膜上的受體作用，而是直接作用在生物膜上形成孔洞。我們使用被廣泛研究的抗菌胜肽-Magainin 2與混合的脂質雙層膜作用，研究脂質成分對孔洞形成門檻濃度((P/L)*)的影響。混合脂質雙層膜的成份為中性的脂質分子，Di(22:1)PC，混合不同比例的帶負電脂質分子，DOPS。對三種不同PC/PS比例的脂質雙層膜，利用指向性圓二色光譜(OCD)和多片層X光繞射(LXD)量測一系列不同magainin 2對脂質分子莫耳比(P/L) 的樣品，。
我們用OCD偵測α螺旋抗菌胜肽在膜上的方位，並進一步的決定孔洞形成的門檻濃度(P/L)*。同樣的樣品亦使用LXD來決定脂質雙層膜的結構來得到雙層膜的厚度。OCD實驗結果顯示，在低濃度時Magainin 2吸附在膜面上，當濃度超過門檻濃度時，部份的Magainin 2會改變方位插入膜中形成孔洞。LXD實驗結果顯示，脂質雙層膜的厚度會隨著P/L增加而線性變薄，該實驗數據可以得到單一個Magainin 2吸附膜面所造成的面積改變(AP)。最後，我們利用建立在生物膜彈性體理論上的兩態模型來分析實驗結果，得到與孔洞形成相關的各個參數值。
同時文中我們提出脂質重新分佈的假說來解釋實驗結果，當Magainin 2在均勻混合Di(22:1)PC與DOPS的脂膜系統時，DOPS會重新排列吸附在抗菌胜肽的周圍不再均勻分佈。除此以外，我們會比較不同脂質雙層膜組成中的門檻濃度和其中的參數值，並討論不同混合比例的脂質對Magainin 2形成孔洞的影響，進一步了解抗菌胜肽與脂膜的作用機制。結果顯示，脂質雙層膜中帶電脂質的比例與Magainin 2形成孔洞所需的門檻濃度無相關性。

摘要(英)

Antimicrobial peptides in the biological innate immune system are the important weapons to kill invading microbes. They are able to bind to membrane directly to forming pore in the membrane rather than the receptor. We used the well-studied antimicrobial peptide, Magainin 2, as a model peptide to interact with mixed lipids bilayer to study the correlation between the lipid composition and threshold concentration of forming pore ((P/L)*).The neutral lipid, Di(22:1)PC, was mixed with negative charged lipid, DOPS, in different molar ratio to produce the mixed lipid bilayers. For each PC/PS mixed ratio, a series of samples of different peptide-to-lipid molar ratios (P/L) were measured by Oriented Circular Dichroism (OCD) and Lamellar X-ray Diffraction (LXD).
The Oriented Circular Dichroism (OCD) was used to probe the peptide orientation on the bilayer as well as determine the threshold concentration ((P/L)*). The samples with the same P/L were measured by Lamellar X-ray Diffraction (LXD) to determine not only electron density but also thickness of the bilayer. OCD results show that magainin 2 bind on the bilayer surface in the low P/L region. When P/L exceeds threshold concentration (P/L)*, a part of peptides changed their orientation to insert into the bilayer. The LXD results show that the bilayer thickness linearly decreases with the P/L increasing. The area expansion induced by one magainin 2 binding on membrane surface (AP) were be obtained from X-ray data. Finally, we extracted the parameters of pore formation from OCD and X-ray data by the two-state model based on elastic theory.
In this study, we proposed a “lipid redistribution” hypothesis to explain our data. Instead of uniform distribution of DOPS in the Di(22:1)PC lipid bilayer, the DOPS lipids will aggregate around the peptides when peptides are added into lipid bilayer. Otherwise, we also compared the threshold concentrations and these parameters in different mixed lipid bilayers and discussed the effects of different compositions to understand the mechanism of magainin2-membrane interaction. The result shows that there is no correlation between threshold concentration and the ratio of charged lipids.

關鍵字(中)

★ 指向性圓二色光譜
★ 多片層X光繞射
★ 抗菌胜肽
★ 電荷效應

關鍵字(英)

★ Magainin 2
★ OCD
★ LXD

論文目次

中文摘要………………………………………………………………………………………………………………………………………i
英文摘要……………………………………………………………………………………………………………………………………ii
誌謝……………………………………………………………………………………………………………………………………………iii
目錄……………………………………………………………………………………………………………………………………………iv
圖目錄…………………………………………………………………………………………………………………………………………vi
表目錄………………………………………………………………………………………………………………………………………vii
符號與縮寫說明 …………………………………………………………………………………………………………………viii
一、前言　……………………………………………………………………………………………………………………………………1
1-1抗菌胜肽……………………………………………………………………………………………………………………………… 1
1-2生物膜組成……………………………………………………………………………………………………………………………3
1-3抗菌胜肽與生物膜交互作用………………………………………………………………………………………………7
1-4實驗目的………………………………………………………………………………………………………………………………11
二、材料與方法　……………………………………………………………………………………………………………………13
2-1 實驗材料 …………………………………………………………………………………………………………………………13
2-2 實驗樣品製備 …………………………………………………………………………………………………………………15
2-3實驗儀器………………………………………………………………………………………………………………………………18
　2-3-1指向性圓二色光譜儀(Oriented Circular Dichroism, OCD)……18
　2-3-2多片層X-光繞射(Lamellar X-ray Diffraction, LXD)………………22
三、數據分析結果……………………………………………………………………………………………………………………27
3-1生物膜彈性理論………………………………………………………………………………………………………………27
3-1-1抗菌胜肽吸附在膜上的自由能變化…………………………………………………………………………27
3-1-2抗菌胜肽吸附膜面與插入膜中形成孔洞兩者共存態的自由能變化…………………28
3-2指向性圓二色光譜實驗結………………………………………………………………………………………………29
3-2-1數據分析 …………………………………………………………………………………………………………………29
3-2-2 結果 ………………………………………………………………………………………………………………………32
3-3多片層X光繞射實驗結果………………………………………………………………………………………………36
3-3-1 數據分析 ………………………………………………………………………………………………………… 36
3-3-2 結果 ……………………………………………………………………………………………………………39
3-4數據統整……………………………………………………………………………………………………………………………47
四、結果與討論……………………………………………………………………………………………………………………48
4-1親油區厚度(h) ……………………………………………………………………………………………………48
4-2單一抗菌胜肽所造成的膜面積增加(AP)…………………………………………………………………50
4-2-1 Magainin 2和Melittin的AP ……………………………………………………………51
4-2-2加入DOPS帶電脂質對AP的影響…………………………………………………………………………52
4-2-3加入DOPS和DOPG的AP差別…………………………………………………………………………………55
4-3單一抗菌胜肽插入膜中造成的面積變化(βAP)………………………………………………………57
4-4抗菌胜肽表面吸附態與垂直插入態的結合能差(Δε)……………………………………………58
4-5孔洞形成門檻濃度(P/L)*…………………………………………………………………………………………60
五、結論…………………………………………………………………………………………………………………………………61
參考文獻……………………………………………………………………………………………………………………………………63

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指導教授

李明道(Ming-Tao Lee)

審核日期

2013-7-19

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