利用拉曼光譜檢測支撐式細胞膜中膜蛋白之結構變化

Abstract

作為溝通細胞內外的媒介，膜蛋白參與了許多細胞的生理作用如物質傳輸、受 體結合與訊息傳遞。為了研究膜蛋白的功能，解析膜蛋白結構便成為了首要目標。 目前常用於研究蛋白結構的方法包含X-ray晶體學、蛋白質核磁共振 (NMR) 以及 冷凍式電子顯微鏡 (cryo-EM) 等技術。然而，X-ray晶體學及冷凍式電子顯微鏡無 法在水溶液環境中研究蛋白質的動態變化，而核磁共振雖可達成前述研究，其可分 析的蛋白質大小卻受限於 25kDa 以下。為了突破這些困境，我們利用了共軛焦拉 曼光譜儀來研究被保存在支撐式細胞膜中的膜蛋白，並且在水溶液的環境下檢測 膜蛋白的動態結構變化。我們使用化學發泡技術來從具有大量表達硝酸鹽運輸蛋 白 (CHL1) 的青蛙卵細胞中取得含有該蛋白的巨大細胞囊泡，並將其在基材上形 成支撐式細胞膜。同時，我們也運用了金奈米增強來自膜蛋白的微弱拉曼光譜訊號， 並觀察到硝酸鹽運輸蛋白在不同磷酸鹽濃度環境中 amide I 和 amide III 區域的 特徵峰變化，發現其與硝酸鹽運輸蛋白的磷酸化相關。透過曲線擬合 (curve-fitting) 分析，我們進一步將這些特徵峰變化與蛋白結構變化之間的關聯做出解讀。這些結 果顯示我們所發展的技術不僅提供了非侵入、無標記式研究生物材料的方法，也在 研究膜蛋白結構變化方面具有巨大潛力。

Membrane proteins involve in several cellular processes such as substrate binding, signal transduction and ion transport. However, it is difficult to directly study membrane proteins in native cells due to the complexity of the cell membrane. Although there are some well-developed techniques, such as X-ray crystallography and cryo-electron microscopy (cryo-EM), for studying membrane protein structure, it is still challenging to study the protein dynamic changes after stimulation. In this study, we used confocal Raman spectroscopy to study membrane proteins embedded in supported cell membranes and examined the dynamic changes of protein structures. In order to study the conformational changes of a nitrate transporter, CHL1, we first derived giant plasma membrane vesicles from CHL1-injected Xenopus oocytes and formed supported cell membrane platforms. We also used gold nanoparticles to enhance the weak Raman signals originated from our samples and observed the protein characteristic peak changes in amide I and amide III regions. We found the correlation between the Raman peak changes and the CHL1 phosphorylated state, and interpreted how the peak changes might be correlated to the protein conformational changes. The results show that this technique provides a non-invasive and label-free way to study biomaterials in the native environment and has a great potential in studying conformational changes of membrane proteins.