利用指向性圓二色光譜及多片層X光繞射技術研究抗菌胜肽與細胞膜作用的電荷效應

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謝明桓(Ming-Huan Hsieh) 查詢紙本館藏

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生物物理研究所

論文名稱

利用指向性圓二色光譜及多片層X光繞射技術研究抗菌胜肽與細胞膜作用的電荷效應
(Studying the charge effect on peptide-membrane interaction by oriented circular dichroism and lamellar X-ray diffraction)

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摘要(中)

抗菌胜肽在生命體的先天防禦系統中扮演著重要角色。目前已知抗菌胜肽可藉由在細胞膜上形成孔洞來造成微生物死亡。近年來因為抗生素的濫用，造成許多微生物產生抗藥性。因此研究抗菌胜肽可幫助克服微生物產生抗藥性的問題。
目前已知抗菌胜肽從水溶液中到膜面上形成孔洞有兩步驟：(1)由於抗菌胜肽本身帶有正電荷，會受到微生物細胞上帶有的脂多醣、肽聚醣等帶有負電荷的物質吸引而接近膜面近而吸附至膜面上，(2)當吸附上膜面的胜肽與脂質分子的濃度(P/L)達到門檻濃度((P/L)*)，胜肽將插入膜面，進而行成孔洞。目前已有不少微脂粒的螢光實驗及恆溫滴定微卡計來研究步驟(1)中的電荷效應，但抗菌胜肽吸附至脂膜後的電荷效應尚未被研究完全，本文中利用指向性圓二色光譜儀及多片層X光繞射技術來研究抗菌胜肽吸附至膜面上後，所形成孔洞所需之門檻濃度及抗菌胜肽造成膜厚度的改變量，並藉由膜的彈性模型來分析數據，藉以瞭解抗菌胜肽與脂膜間的電荷效應。
本實驗的結果顯示電荷效應使得門檻濃度稍微的提高，代表胜肽與膜的電荷效應主要作用在步驟(1)。在水溶液中，靜電作用將使得帶有正電的胜肽聚集在帶有負電的膜附近。因此，就算門檻濃度提高，膜附近的胜肽濃度也夠高，可輕易地跨越門檻，進而形成孔洞。

摘要(英)

Antimicrobial peptides plays an important role in the innate defense systems of animals and plants. Antimicrobial peptides are known to kill the invading microbes by forming pores in the membrane. Recent years, many microbes exhibit drug resistances due to the overuse of antibiotics. The investigation on antimicrobial peptides should provide the solution to overcome the drug resistance.
There are two steps in the process of antimicrobial peptides binding to membrane to form pores (1) In aqueous solution, the antimicrobial peptides will be attracted by negative charged membranes composed of charged lipids (such as: lipopolysaccharide, peptidoglycan, etc. ) and accumulate around the membrane. Then the peptides will bind to the membrane surface due to hydrophobic interaction. (2) When the concentration of binding peptides excess the threshold concentration ((P / L) *), a part of peptides will change their state to insert into the membrane and form pores. Currently, many fluorescence leakage and isothermal titration calorimetry (ITC) experiments study the charge effect on the step (1), but the charge effect on the pore formation induced by antimicrobial peptide binding to the membrane has not been clarified. In this thesis, Oriented circular dichroism (OCD) is used to determined the threshold concentration (P/L)* of pore formation and lamellar-lamellar X-ray diffraction (LXD) is used to probe the thinning of membrane thickness due to peptides binding. We used the free energy model based on elastic theory to analyze the data to understand the charge effect on peptide-membrane interaction.
The result shows that the threshold concentration becomes slightly higher due to charge effect. That means the charge effect on peptide-membrane interaction in solution is dominated by step (1). In aqueous solution, the electrostatic interaction will force positive charged peptides to acuminate around the negative charged membrane. Consequently, the local concentration of peptides is high enough to excess the threshold easily even if the threshold is slightly higher.

關鍵字(中)

★ 胜肽
★ 細胞膜
★ 指向性圓二色光譜
★ 多片層X光繞射技術
★ 電荷效應

關鍵字(英)

★ peptide
★ membrane
★ oriented circular dichroism
★ lamellar X-ray diffraction
★ charge effect

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……………………………………………………………………………………………………………………… viii
符號說明…………………………………………………………………………………………………………………… ix
一、前言………………………………………………………………………………………… 1
1-1 細胞膜…………………………………………………………………………………………………… 1
1-2 抗菌胜肽……………………………………………………………………………………………… 3
1-2-1 抗菌胜肽結構分類…………………………………………………………………………… 3
1-2-2 抗菌胜肽與細胞作用機制之相關模式………………………………………… 4
1-3 陽離子抗菌胜肽與生物膜作用………………………………………………………… 7
1-4 陽離子抗菌胜肽與生物膜研究………………………………………………………… 7
1-5 實驗目的……………………………………………………………………………………………… 8
二、材料與方法……………………………………………………………………………… 11
2-1 實驗材料……………………………………………………………………………………………… 11
2-2 實驗樣品製備……………………………………………………………………………………… 11
2-3 實驗儀器………………………………………………………………………………………………… 12
2-4 指向性圓二色光譜儀…………………………………………………………………………… 12
2-4-1 圓二色光譜儀……………………………………………………………………………………… 12
2-4-2 抗菌胜肽的圓二色光譜……………………………………………………………………… 13
2-4-3 指向性圓二色光譜……………………………………………………………………………… 14
2-4-4 OCD實驗方法………………………………………………………………………………………… 15
2-4-5 OCD數據分析與處理…………………………………………………………………………… 18
2-5 多片層X光繞射………………………………………………………………………………………… 20
2-5-1 LXD實驗原理與分析…………………………………………………………………………… 20
2-5-2 LXD實驗方法………………………………………………………………………………………… 24
三、實驗結果與討論……………………………………………… 26
3-1 生物膜之彈性模型…………………………………………………………………………………… 26
3-2 實驗結果……………………………………………………………………………………………………… 27
3-2-1 指向性圓二色光譜結果………………………………………………………………………… 27
3-2-2 多片層X-光繞射結果…………………………………………………………………………… 31
3-3 討論……………………………………………………………………………………………………………… 37
3-3-1 頭區較小的脂膜厚度……………………………………………………………………………… 37
3-3-2 單一胜肽吸附膜面所造成膜表面積的變化量隨DOPG分子的變化 38
3-3-3 單一胜肽插入膜面所造成膜表面積的變化量隨DOPG分子的變化 39
3-3-4 胜肽吸附膜面與插入膜內結合能差…………………………………………………… 41
3-3-5 門檻濃度隨DOPG分子變化…………………………………………………………………… 42
四、結論……………………………………………………………………………………… 44
參考文獻…………………………………………………………………………………………………………………………… 45

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指導教授

李明道(Ming-Tao Lee)

審核日期

2012-1-31

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