精準調控的脂質環境中細菌視紫質之研究

Study of Bacteriorhodopsin in a Controlled Lipid Environment

葉純青(Vivien Yeh)

指導教授 ： 陳振中

共同指導教授 ： 余慈顏(Tsyr-Yan Yu)

國立臺灣大學/理學院/化學研究所/博士(2017年)

https://doi.org/10.6342/NTU201702808

摘要

膜蛋白的結構與功能會隨著細胞膜的環境而改變，因此模擬細胞膜環境是膜蛋白研究中相當重要的課題。脂質奈米碟的組成是由兩條平行的膜支架蛋白環狀包住磷脂質的疏水端，以形成穩定的雙層細胞膜結構。將膜蛋白鑲嵌於脂質奈米碟可以模擬原始細胞膜的雙層脂環境，穩定膜蛋白的結構，並使膜蛋白的結構與功能之研究更為容易。利用脂質奈米碟能夠形成精準調控的雙層脂膜的特性，我們證實脂質成分能夠調控膜蛋白的功能。為了觀察不同脂質奈米碟的脂質成分對膜蛋白造成的影響，我們將單體細菌視紫質包入由雙電性與陰電性脂質以不同比例形成的脂質奈米碟中，並利用可見光瞬態吸收光譜來偵測細菌視紫質的光迴圈動力學反應。實驗結果顯示，細菌視脂質的光迴圈速度會隨著脂質奈米碟中帶陰電性親水端的脂質減少而趨緩，光迴圈路徑也有所改變，證實了脂質奈米碟的脂質組成與膜蛋白質子幫浦功能間的關聯性。我們利用光致電流實驗更進一步地發現，脂質不僅影響光迴圈動力學，亦具有幫助質子傳遞的功能，在細菌視紫質質子幫浦的運作中扮演重要的角色。為了探討脂質奈米碟的尺寸對膜蛋白造成的影響，我們透過改變膜支架蛋白的長度來組成不同大小的脂質奈米碟。相較於在界面活性劑微胞環境中，被包入脂質奈米碟的細菌視紫質有明顯不同的光迴圈動力學。但是，細菌視紫質的光迴圈速度隨著奈米碟的尺寸並沒有顯著的改變。這顯示，相較於脂質成分的影響，脂質奈米碟大小對膜蛋白的功能並沒有顯著的影響。

綜合以上結果，我們證實了脂質奈米碟的成分和大小與嵌於其上膜蛋白之功能相關性。我們更進一步地研究能直接將膜蛋白從生物細胞膜上轉移至脂質奈米碟中的方法。利用特別設計的環狀膜支架蛋白，我們將三聚體細菌視紫質與部分脂雙層從古生嗜鹽桿菌的原生紫膜上直接轉移至脂質奈米碟中，在此步驟中無需添加人工合成脂質或脂質萃取物。利用此一方法獲得的原生紫膜奈米碟透過可見光圓二色譜與可見光瞬態吸收光譜證實細菌視紫質可保有原始的形式與功能。原生脂質奈米碟的圓形型態是以未負染高解析透射電子顯微鏡影像觀察而得，而紫膜奈米碟的脂質成分則是經由核磁共振光譜儀來分析。我們成功地製備含有原生脂雙層與膜蛋白的脂質奈米碟，保留了膜蛋白原生的環境而使膜蛋白的研究更接近生物體中的狀態。

關鍵字

膜蛋白 ； 脂質奈米碟 ； 細菌視紫質 ； 光迴圈動力學 ； 可見光瞬態吸收光譜 ； 脂質組成成分 ； 生物原生膜

並列摘要

The structure and function of membrane protein often shows dependency to the membrane environment. Monodisperse lipid nanodisc has been a useful tool for mimicking membrane environment for the biophysical characterization of membrane proteins. Nanodisc is a high-density lipoprotein with a disk shaped core of lipid molecules, wrapped by two copies of membrane scaffold protein, providing membrane protein a near-native lipid bilayer environment while suitable for spectroscopic studies. By incorporating monomeric bacteriorhodopsin (bR) into nanodisc, we demonstrate how to manually manipulate the function of membrane protein by altering the property of the nanodisc, such as lipid composition and the size of nanodisc. Embedding in nanodisc composed of different ratios of synthetic zwitterionic lipid to negatively charged lipid, the photocycle kinetics of bacteriorhodopsin was found to alter as the lipid composition changed. Full wavelength transient absorption spectroscopy revealed that as the content of PG decreased, the duration of the photocycle of bR increased drastically and the photocycle pathway was altered. Further measurement using transient photocurrent indicated that lipid molecule not only affects the photocycle kinetics but also plays a role in the release of proton from the protein to the bulk solution during proton translocation. To study the effect of the size of nanodisc, E. coli expressed bR was incorporated into nanodisc of two different sizes. The photocycle kinetics of bR embedded in nanodisc was compared to bR in detergent micelle, and was shown to have significant different photocycle kinetics. However, the sizes of nanodisc were found to exhibit no significant effect on the function of bR embedded.

While demonstrating how the synthetic membrane environment can influence the function of membrane protein, we further improved the native environment by using a novel one-step method to incorporate trimeric bR into lipid nanodisc prepared from the native purple membrane of Halobacterium salinarum, without adding any synthetic lipid or lipid extracts. The method was demonstrated to produce homogenous sample with sufficiently high yield suitable for biophysical studies. The trimeric conformation of bR was verified using visible wavelength circular dichroism, and Zernike phase TEM image showed a circular disk like morphology. The lipid composition of the native purple membrane nanodisc was investigated with 31P NMR and liquid chromatography mass spectrometry, where the essential lipids are shown to be maintained. Lastly, the preservation of photocycle activity was confirmed using transient absorption spectroscopy. We demonstrated the feasibility of transferring membrane protein into nanodisc directly from the native membrane, surrounded by native lipid molecules to preserve the biological structure.

並列關鍵字

Membrane protein ； lipid nanodisc ； bacteriorhodopsin ； photocycle kinetics ； transient absorption spectroscopy ； lipid composition ； native membrane

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