利用拉曼光譜來檢測於支撐式脂質膜中之脂質以及膜蛋白

Abstract

膜蛋白在細胞內扮演著重要角色，但目前其研究仍受限於缺乏適當的工具來使膜蛋白在其自然狀態中被觀測其結構及功能。傳統方法通常需要添加清潔劑來從細胞膜萃取出膜蛋白做後續觀測以及研究，然而這樣的方法常被詬病可能會破壞其功能或結構。為了突破這個困境，我們利用共軛焦拉曼光譜儀來對在支撐式細胞膜平台中的脂質和膜蛋白進行研究。我們利用石墨烯增強脂雙層和膜蛋白微弱的拉曼光譜訊號。但因為石墨烯存在消光效應，我們利用光蝕刻得到孔洞圖案以實現其偵測和觀察。首先我們應用拉曼光譜來研究腎結石疾病中最常見的草酸鈣（CaOx）結晶如何保留在脂質膜上。我們製備了具有不同膜流動性的人工脂質膜，發現附著在具有高流動性的脂質膜上的晶體會誘導其下脂質膜重新排列，並增強晶體附著。拉曼光譜結果顯示了脂質膜發生了從流體相到凝膠相的相變。而此由晶體誘導之相變可能使得脂質分子可以匹配晶格排列，降低系統能量，進而顯著增強晶體附著。此外，我們運用發泡法從青蛙卵細胞獲得了具有硝酸鹽運輸蛋白(CHL1)的巨型單層囊泡，並在石墨烯上形成了支撐式細胞膜平台。我們觀察到硝酸鹽運輸蛋白Amide III特徵峰的變化，並發現其與硝酸鹽運輸蛋白的磷酸化相關，我們也對這些特徵峰變化和結構變化之間的連結做出解讀。我們的結果顯示此技術不僅提供了一種非侵入性研究生物材料的新方法，也在研究膜蛋白結構變化方面具有巨大潛力。

While membrane proteins play important roles in various cellular processes, there are still limited tools and techniques to study them. The traditional method is to disrupt cell membranes by adding detergents to extract the membrane proteins for further study. However, the method usually would disrupt the function or structure of the membrane proteins. In this study, we used confocal Raman spectroscopy to study supported lipid membranes and the embedded membrane proteins. Our analytical technique enables us to obtain a precise focal plane so that we can obtain robust Raman signals from a few nanometer-thick supported lipid membranes. The weak Raman signals were enhanced with graphene. We used the photolithography method to pattern graphene so that we can observe the location of the cell membrane in spite of the fluorescence quenching effect of graphene. We first applied Raman spectroscopy to examine the retention mechanism of calcium oxalate (CaOx) crystals on lipid membranes, which is related to kidney stone diseases. We prepared lipid membranes with different membrane fluidity and found that crystal formation on the lipid membrane with high fluidity can induce phase transition. The Raman result suggests that the crystal may induce phase transition of lipid membrane from the liquid phase to the gel phase which results in lipid molecules to closely align to match the crystal lattice to reduce the system energy and therefore significantly enhance the crystal attachment. In addition, we also derived giant unilamellar vesicles with nitrate transporter CHL1 from oocytes and formed supported plasma membranes on graphene supports. We observed the characteristic peaks of amide III from CHL1 in the supported plasma membrane, and found the correlation between the spectrum change and the phosphorylation. We also interpreted how the spectrum change may be correlated to the structural change. The results show that this technique not only offers a new way to study biomaterials noninvasively but also has potential in studying changes of protein structure in the native membrane environment.