表面電漿共振感測器觀測固定化蛋白質的構型變化與島狀脂双層膜的建構

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姓名

陳良宇(Liang-Yu Chen) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

表面電漿共振感測器觀測固定化蛋白質的構型變化與島狀脂双層膜的建構
(Biomolecules Interaction Analysis With Biosensors:I. Monitoring the Conformational Changes of Immobilized Proteins and Evaluation of Measurement Uncertainty II. The Fabrication of Mimetic Lipid Bilayer on Sensor Surface and Its Application.)

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摘要(中)

生命系統中許多事件的發生不外乎因為兩個（或更多）分子的相互靠近形成複合物所開始的，這些分子交互作用可以是蛋白質與蛋白質、核酸與蛋白質、核酸與核酸、蛋白質與膜、或化學小分子與生物分子的交互作用。當一個分子與另一個特定分子接觸，所有的動力學(kinetics)、熱力學(thermodynamics)及分子構形配對(conformational matching)等原則都將參與作用，並決定了此交互作用的生物功能。為了能清楚地描述分子交互作用的過程，能夠提供高靈敏、高專一、精確定量、快速與即時資訊的生物感測器是重要的分析工具平台。
本研究以表面電漿共振感測器(surface plasmon resonance sensor)進行生物分子交互作用分析(biomolecules interaction analysis)的應用探討，就資訊之產出而言，固定化分子構形的研究不但是分子辨識的核心課題且最具挑戰性。實驗結果顯示隨著變性劑(GdmCl或urea)濃度的增加，固定化蛋白質(Lysozyme或RNase A)構形將產生變化，SPR角度訊號呈現增加的趨勢，而當還原劑(32 mM DTT)一起作用的情況下，訊號卻呈現減少的變化趨勢。藉由探討表面電漿共振感測器的量測不確定度分析，評估角度量測之訊號組成來源並建立了數據分析準則，我們揭露了溶液、溶質與固定化基材交互作用對觀測的影響。其次，固定化蛋白質受到變性劑影響時構形變化的直接觀測，也確認了許多蛋白質分子結構的重要資訊，如構形的緻密度、疏水性區塊暴露、双硫鍵對結構穩定性的影響等。
另一方面，結合石英振盪微天平的動態監測能力與微觀表面形態分析，探討微脂粒組成成份對吸附動力學機構與膜結構的影響與驗證，得到建構穩定島狀脂双層膜的配方，並以蜂毒蛋白之穿膜過程為例，展示島狀脂双層膜結構在生物物理、生物化學研究上應用的可行性。單一磷脂質 (DPPC)較混合脂質(egg PC)所組成的脂膜具有較大的結構穩定性，也較易形成島狀脂双層膜的結構，而膽固醇的添加可顯著地提高脂膜結構的穩定性並抑制穿膜過程的發生。相信此類生物膜的實驗系統將提供生物感測器更真實與更廣泛應用的生物分子交互作用之界面平台。

摘要(英)

Biological events are initiated by bio-molecular interactions, e.g. protein-protein, protein-nucleic acid, protein-lipid and chemical ligand-receptor interactions. The kinetics, thermodynamics and conformational matching involved in the contact between two or more molecules determine whether the interaction has any biological meaning in the real world. To clearly characterize the process of molecular interactions and further explore their true behaviors in biological systems, highly sensitive, specific, precise and fast biosensors are required to obtain the real-time and quantifiable information of interactions.
In this study, we investigated the application of surface plasmon resonance sensor (SPR) in the bio-molecular interactions analysis. The core of molecular recognition lies on the study on immobilized bio-molecule conformation, which is a very challenging research field.　In the regard of uncertainty analysis of SPR, we evaluated the source and composition of signals of angle measurements and established the principle of data analysis. Based on this, we disclosed the effect of interactions between bulk solution, solute and immobilized matrix on measurement. Further more, the direct observation of conformational changes of immobilized proteins in unfolding and refolding derived a lot of important structural information of protein, such as the compactness of conformations, the exposure of hydrophobic region and the effects of intrinsic disulfide bonds on structural stability of immobilized proteins.
On the other side, the combination of real-time monitoring capacity of QCM and microscopic surface morphology by AFM was employed to investigate the effects of liposome composition on the structure of supported lipid bilayer and the kinetics mechanism of absorption at the solid-liquid interface. The formula of stable vesicular lipid bilayer was established. We also demonstrated the possibility of application of vesicular lipid bilayer in biophysical and biochemical studies by the transmembrane process of melittin. We believe that the mimetic membrane systems would be ideal platform candidates to study bio-molecules interactions in the application of biosensors.

關鍵字(中)

★ 蛋白質固定化
★ 構型變化
★ 島狀脂双層膜
★ 石英振盪微天平
★ 表面電漿共振感測器

關鍵字(英)

★ immobilization protein
★ quartz crystal microbalance
★ biosensor
★ surface plasmon resonance
★ lipid bilayer
★ cconformational changes

論文目次

1. 生物分子交互作用分析 1
1.1 生物分子交互作用與辨識 1
1.2 生物感測器 3
1.3 表面電漿共振感測器 5
1.4 探索生物分子交互作用 8
1.4.1生物交互作用分析之定量原則 10
1.4.2專一性分析 11
1.4.3 動態機構分析 12
1.5如何建構具有生物功能的感測界面 14
1.5.1共價結合修飾界面的固定化 15
1.5.2非共價性之親合性捕捉技術 16
1.5.3 擬生物膜系統的建構 17
1.6 SPR-BIA的應用 19
1.6.1生物性材料設計與工程應用 19
1.6.2生物晶片的檢測 20
1.6.3食品安全的檢測 21
1.6.4藥物開發 21
1.6.5疾病與基礎生化的研究 22
1.6.6配基的釣取 (Ligand Fishing) 24
1.6.7固定化生物分子構型 24
1.7 運用生物感測器探討生物分子交互作用的分析準則 25
2. PART A：運用SPR觀測固定化蛋白質構形的變化 29
2.1實驗材料與方法 29
2.1.1藥品及試劑 29
2.1.2自組式表面電漿共振儀 30
2.1.3晶片表面改質與蛋白質的固定化 32
2.1.4運用SPR觀測固定化蛋白質分子構形的變化 32
2.1.5變性劑對溶液中蛋白質構形的影響 33
2.2 結果與討論 34
2.2.1 溶劑與基材交互作用影響SPR觀測之量測不確定度 34
2.2.2 探討變性劑對固定化蛋白質構形的影響 38
2.3 總結：運用SPR觀測固定化蛋白質之構形變化 44
3. PART B：建構擬生物膜之BIA系統 45
3.1 實驗材料與方法 45
3.1.1藥品及試劑 45
3.1.2微脂粒的製備 45
3.1.3 感測器金屬界面的修飾 46
3.1.4 脂双膜的固定化與表面形態分析 46
3.1.5 脂双膜的固定化與即時觀測 47
3.2 結果 48
3.2.1 固定化脂双層膜的形態分析 48
3.2.2 脂双層膜吸附的動態觀測與定量資訊 48
3.3 討論：建構脂双層膜之動力學機構分析 50
3.4 應用範例：島狀脂双層膜與蜂毒蛋白之穿膜交互作用 54
3.4.1溶液系統下蜂毒蛋白與微脂粒之交互作用 54
3.4.2擬生物脂双層膜系統與蜂毒蛋白交互作用之形態分析 55
3.4.3蜂毒蛋白穿膜作用之動力學機構分析 56
3.5 總結：擬生物膜的建構 59
4. 總結 60
5. 參考文獻 62
附錄 82
1. Effects of solute–matrix interaction on monitoring the conformational changes of immobilized proteins by surface plasmon resonance sensor (2005) Talanta, In press. [82-87]
2. Thermodynamics and mechanism of ssDNA hybridization below the melting temperature by isothermal titration calorimetry (2005) Thermochimica Acta, 433: 83-87. [88-92]
3. The effects of denaturants on protein conformation and behavior at air/solution interface (2005) Colloids and Surfaces B: Biointerfaces, 41: 1–6. [93-98]
4. 重要文獻資料之摘要整理 (補充) [99-105]

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2005-7-20

推文