使用指向性圓二色光譜與多片層X光繞射技術進行daptomycin與細胞膜作用之結構研究

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張原榜(Yuan-Pang Chang) 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與多片層X光繞射技術進行daptomycin與細胞膜作用之結構研究
(Structural studies on the interaction between daptomycin and membranes by oriented circular dichroism and lamellar X-ray diffraction)

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摘要(中)

抗菌胜肽存在於動植物的免疫系統中，藉由直接與細胞膜作用來消滅入侵體內的病原體或微生物，而細胞膜的組成很難因基因突變發生變化，所以不易產生抗藥性，因此研究抗菌胜肽的抑菌機制對解決日益嚴重的抗藥性問題是一個重要的課題。

daptomycin為環脂肽，是獲得美國FDA認證的新型結構抗菌胜肽。它會與革蘭陽氏菌的細胞膜作用導致膜通透來殺死細胞，在有鈣離子存在與細胞膜上含有phosphatidylglycerol (PG)脂質分子的情況下才具有抑菌活性。儘管經過多年的臨床使用與研究，其具體的分子作用機制仍是未知的。
此篇研究中，我們使用指向性圓二色光譜(Oriented Circular Dichroism, OCD)來測量daptomycin在細胞膜上的二級結構的變化，進一步決定其與細胞膜的結合狀態。再使用多片層X光繞射(Lamellar X-ray Diffraction, LXD)測量daptomycin與細胞膜結合後所產生的厚度變化。

OCD的結果顯示，在PG與鈣離子都存在的情況下，daptomycin的OCD光譜在232(nm)附近會有明顯翻轉，其光譜翻轉情形與daptomycin和鈣離子濃度有關。藉由分析其光譜翻轉，發現與細胞膜結合的daptomycin超過臨界濃度，daptomycin會改變其與細胞膜的結合狀態；且確認了daptomycin與鈣離子的化學劑量比例約為1到2之間。LXD的結果顯示，與daptomycin作用會使細胞膜變薄，而與鈣離子作用會使細胞膜變厚。在daptomycin、PG與鈣離子並存的情況下，細胞膜則會變厚，我們提出不同模型進行計算來解釋細胞膜厚度的變化，進一步討論daptomycin、鈣離子與細胞膜的作用。

摘要(英)

Antimicrobial peptides are widely used by animals and plants in their innate immune systems to eliminate invading pathogens or microbes via directly targeting to their membranes. The composition of the membrane is difficult to be changed by gene mutation so that it is rare to exhibit antibiotic resistance. Therefore, studies on the antibacterial mechanism of antibacterial peptides is an key issue to solve the serious problem of antibiotic resistance.
Daptomycin, a cyclic lipopeptide, represents a new structural class of the FDA approved antibiotics. It interacts with the cytoplasmic membranes of Gram-positive pathogens causing membrane permeabilization to kill cells. The antibiotic activity is calcium ion dependent and correlates with the targeted membrane’s content of phosphatidylglycerol (PG), otherwise its underlying molecular mechanism is so far unknown in despite of clinical usages and researches for many years. Here we used oriented circular dichroism (OCD) to probe the change of second structure to determine the binding states of daptomycin in membranes. Lamellar X-ray diffraction (LXD) was used to monitor the thickness change of membrane induced by daptomycin binding.
In the coexistence of daptomycin, PG and calcium ions, the result shows that OCD spectra of daptomycin will be significantly reversed around 232 (nm) and the reversed spectra is correlated to the concentrations of daptomycin as well as calcium ions. Consequently, it indicates that the concentration of binding daptomycin excess a threshold, daptomycin will change its state. And the stoichiometric ratio of daptomycin to calcium was confirmed between 1 and 2. The LXD result shows that daptomycin induced membrane thinning and calcium ions induced membrane thickening, respectively. In the coexistence of daptomycin, PG and calcium ions, membrane thickening was observed. In this study, we attempt to propose different models to fit experimental data to clarify the change of membrane thickness in different concentrations of daptomycin and calcium ions. Finally, the daptomycin/calcium ion/membrane interaction will be discussed.

關鍵字(中)

★ 抗菌胜肽

關鍵字(英)

★ AMP

論文目次

目錄
中文摘要 i
英文摘要 ii
誌謝 iii
目錄 iv
圖目錄 vi
表目錄 viii
符號與縮寫說明 ix
一、前言 1
1-1 細胞膜 1
1-2 抗菌胜肽 4
1-3 daptomycin 5
1-4 實驗目的 8
二、材料與實驗方法 9
2-1 實驗材料 9
2-2 樣品製備 11
2-3 實驗儀器 15
2-4 圓二色光譜儀(Circular dichroism, CD) 15
2-5 指向性圓二色光譜(Oriented Circular dichroism, OCD) 15
2-5-1 實驗步驟 17
2-6 多片層X光繞射技術(Lamellar X-ray Diffraction, LXD) 19
2-6-1 LXD 實驗原理與分析 19
2-6-2 LXD實驗方法 22
三、分析與討論 24
3-1 指向性圓二色光譜(OCD)結果分析與討論 24
3-1-1 數據處理 24
3-1-2 比較CD與OCD的結果 27
3-1-3 不同daptomycin與鈣離子濃度的OCD結果 28
3-1-4 分析實驗數據 31
3-2 多片層X光繞射實驗結果分析與討論 35
3-2-1 數據處理 35
3-2-2 daptomycin與脂質雙層膜的作用 45
3-2-3 鈣離子與脂質雙層膜的作用 45
3-2-4 daptomycin、鈣離子與DOPC/PG(7/3)脂質雙層膜的作用 46
3-2-5 模型 I 46
3-2-6 模型 II 49
3-2-7 模型 III 52
四、結論 56
參考文獻 58

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指導教授

李明道(Ming-Tao Lee)

審核日期

2020-7-23

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