利用支撐性脂雙層膜探討細胞膜和蛋白質交互作用及其應用

Supported Lipid Bilayer Model for the Study on Lipid-Protein Interactions and its Applications in Protein Separation Analysis and Purification

郭廷洋(Ting-Yang Kuo)

指導教授 ： 何佳安 ； 陳益佳

國立清華大學/理學院/化學系/博士(2013年)

摘要

由於細胞膜的物理及化學性質變化在生理上扮演重要角色，因此本論文研究除了以支撐性脂雙層(supported lipid bilayer，SLB，將微脂球平鋪於基材上所形成)當作細胞膜之研究模式，藉以探索生物膜的物理化學性質外；亦結合奈米粒子，設計出具有高應用價值的生物探針。
本論文分三大部分：首先在矽晶片上鋪設支撐性脂雙層，並以雙偏極化干涉儀分析之。藉由晶片的干涉條紋變化量可獲得折射率、質量、厚度、雙折射值(birefrigence)等參數，進一步探討脂質膜形成的機制和其抗蛋白質吸附的性質。本部分的研究結果結果除了發現小單層微脂球(small unilamellar vesicles，SUV)適合製備脂雙層外，螢光蛋白質脫附實驗證實脂雙層的抗吸附特性。而應用螢光漂白回復分析(fluorescence recovery after photobleaching，FRAP)則可以證實塗佈在矽材上脂雙層之完整性。若將支撐性脂雙層結合毛細管電泳分析法，可成功分離多種鹼性蛋白質，而未塗佈SLB之毛細管則無法有效分離在中性環境下帶正電的鹼性蛋候白質。
本論文的第二、三部分，針對胞膜的特殊物理現象-脂質筏進行深入探討。由於不同脂質分子間的不互溶現象會造成脂質筏微區域聚集，該現象被發現與訊息傳導和疾病起源關係密切。本論文研究嘗試在矽基材及金奈米粒子上建構具有脂質筏結構的脂雙層，並此仿生基材運用在蛋白質純化和生化分析等領域。
實驗中，使用螢光標記蛋白質和雙偏極化干涉儀進行脂質筏吸附特性的量測，結果證實具脂質筏結構比不具脂質筏結構能多將近3倍的蛋白質吸附量，且其對蛋白質的解離常數(apparent dissociation constant, KD)低約25倍；而吸附在脂質筏上之蛋白質活性，相較於直接吸附於未修飾脂質之矽球表面，則較高。脂質筏結構可藉由簡單升降溫轉換成不具脂質筏結構，進而使已吸附之蛋白質從矽球上脫附。利用脂質筏特性做為蛋白質吸附材料提供一新穎蛋白質純化技術平台。此外，脂質筏提供蛋白質吸附的能力其實是一動態反應的過程，以雙偏極化干涉儀量測蛋白質和脂質筏之吸附親和力，亦發現脂質筏於蛋白質吸附後產生動態擴增的現象。
論文的第三部分，研究目標在於開發合成脂質包覆金奈米粒子之方法，由於此一脂質包覆金奈米粒子具有良好分散性及抵抗高鹽環境或界面活性劑所引發之聚集現象，因此十分適合將此脂質包覆金奈米粒子應用在生化分析。由於過去研究指出，醣脂質(GM1)與花生凝集素(peanut agglutinin lectin，PNA)間存在有選擇性親合結合的能力；因此在脂質配方中摻入GM1，即可利用本論文所設計合成出的金奈米粒子，針對PNA進行快速且高選擇性的偵測，偵測極限可達到5 nM。研究中藉由金奈米粒子系統可以提供量測脂雙層膜上配位基與受體間之交互作用關係的功能。此外，研究中亦觀察到，由於立體組態關係(3D vs. plannar surface)可凸顯多價力效應；舖佈在金奈米粒子上的脂雙層(SLB)中所含的醣脂質(GM1)比平板式脂雙層中的醣脂質具有針對花生凝集素更好的親和力。而添加膽固醇於脂雙層中則可加強醣脂質筏的形成，也因此增強其對花生凝集素的親和力；藉由原子力顯微鏡觀察可以佐證上述現象。

關鍵字

微脂球 ； 脂雙層 ； 雙偏極化干涉 ； 毛細管電泳 ； 脂質筏 ； 奈米粒子 ； 凝集素 ； 原子力顯微鏡

並列摘要

Under physiological environments, the dynamically physical and chemical process within the cell membrane surfaces serves as indispensable parts of biology signaling pathways. Supported lipid bilayer (SLB), demonstrated as a model of cell membranes, has been widely expanded to prospective applications in bioanalytical chemistry or biotechnology. In addition, combination of SLB and nanoparticles provides a novel design for developing highly applicable functional biomaterials.
In the first section of this dissertation, the formation of SLB and their potential biofunctionality against protein adsorption were investigated by dual polarization interferometry (DPI) and capillary electrophoresis (CE). DPI studies on different formulations of double-chained, zwitterionic phospholipidlipids, allow the process of bilayer formation to be followed in situ and in real time. Furthermore the anti-protein adsorption effect provided by the various formulated SLBs was examined by DPI. In addition, the SLB coatings of the same lipid formulations were subsequently employed in CE experiments as a pseudo-stationary phase for demonstrating more efficient separation of alkaline protein standard mixtures. SLB-assisted CE was found to be capable of separating 4 alkaline proteins. This study demonstrates the applicability of DPI to monitor the process of SLB formation; and our findings, obtained by both DPI and CE, confirm that the presence of the SLB reduced drastically the problematic interactions between cationic, alkaline proteins and the negatively charged silica capillary wall, leading to better recovery and efficient separation of the proteins under investigation.
In the second section, we prepared the biomimetic silica microspheres, lipid raft-presenting silica microspheres, from binary phase of lipid mixtures. The formation of lipid raft on resulted silica spheres led to produce nearly 3-time affinity capacity toward lysozyme than non-raft presenting lipid structure, where charged lipids distribute homogeneously on the membrane. The desorption of adsorbed lysozyme was simply achieved by modulating temperature; it was also found in this study that the catalytical activity of adsorbed lysozyme was significantly preserved. Furthermore, DPI was employed to investigate the affinity of lysozyme toward lipid rafts fabricated on the surface of silica chip. Based on the real-time sensorgram acquired by DPI, it ws revealed that (i) lipid raft was capable of enhancing the affinity toward lysozyme; (ii) the cooperative formation of lipid raft was observed. The biomimetic silica microspheres hold the potential of being integrated with other biotechnological tools, for purifying membrane protein or for exploring the mechanism of lipid rafting.
At last, a facile approach in synthesizing mono-dispersed, lipid-capped gold nanoparticles was demonstrated. Such the nanomaterial, fully coated with lipid bilayer and capable of resisting aggregation in high-salt and detergent-containing solutions, was able to be engaged in the development of a novel sensor for monitoring ligand-receptor interaction on the lipid membrane. Furthermore, such sensor offers the feasibility in studying the interaction kinetics between glycolipid and lectin on lipid membrane as well. To prove of the concept, GM1/PNA pair was chosenas model example, and the lipid-capped gold nanoparticle allowed evaluation of apparent dissociation constant (KD). Results show that 3-D configuration of the gold nanoparticles enhanced the multivalent binding effect of glycolipid GM1 to PNA futher comparing to that of the planner format. The addition of cholesterol led to the enhanced affinity of GM1 toward PNA by several folds, which should be attributed to lipid raft formation. The results obtained by atomic force microscopy (AFM) also confirmed the positive correlation between the cholesterol concentration and the size of lipid raft.

並列關鍵字

liposome ； lipid bilayer ； dual polarization interferometry ； capillary electrophoresis ； nanoparticle ； lipid raft ； lectin ； atomic force microscopy

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