- 學位論文
以新式光電流量測觀點剖析微生物視紫紅質之光驅動離子傳輸功能
New insights of a photocurrent method for functional measurement of light-driven ion transportation in microbial rhodopsins
摘要
水是生命不可或缺之物，而水中的離子對生物分子功能的行使扮演至關重要的角色。細胞透過離子的交換與輸送來維持生命，對於嗜鹽古生菌更是如此；其細胞膜上具有微生物視紫紅質，在光驅動之下，產生離子運送作用。其中氯視紫紅質協助氯離子輸送至胞內維持滲透壓，細菌視紫紅質則是將質子向外運送，使其累積在細胞膜外，促進 ATP 合成酶產生細胞能量 ATP。量測電位訊號為探討離子輸送的常見方法，因此本研究第一部份使用氧化銦錫玻片為電極所構築的光電流偵測系統，探討其離子敏感性以及生物分子的適用性。結果顯示本裝置對於質子具有高度專一性並可在廣泛的 pH 範圍內維持信號之高度線性關係，且可辨別光照下細胞膜上目標蛋白質運送質子的流向。藉此方法已可篩選到潛在質子傳輸蛋白質，並可以訊號波形協助研究菌種差異。第二部份使用第一部份之光電流偵測系統，嘗試分析生物中未解之謎。因鹽方扁平古菌 (Haloquadratum walsbyi) 被認為同時具有兩個不同的質子泵細菌視紫紅質，分別為 HwBR 和HwMR，但未能被證實。此部分研究以 E. coli 表達這兩個蛋白質，配合質子傳輸路徑關鍵氨基酸的點突變，在全細胞與蛋白質層次，探討光照下其二者質子運輸所造成之外界 pH 值變化、紫外光/可見光吸收光譜以及光電流訊號差異。以兩個質子泵最重要的保守胺基酸突變來研究，結果顯示 HwBR 為細菌視紫紅質，其 D93負責質子向外輸送，D104 主導質子回收。但 HwMR 僅具有微弱的質子傳輸能力，其 D84N 突變對質子釋出無顯著影響，D95N 突變則會阻撓質子向外傳輸。HwBR 與 HwMR 的特徵吸收光波段相差 55 nm，且後者微弱的質子傳輸能力與非典型的質子傳輸路徑，推測其可能扮演調控訊號傳遞之角色。總結兩大部份研究，以 ITO 為基材之光電流偵測裝置，適合篩選具質子傳輸能力的蛋白質，並可用於分析質子傳輸路徑。
並列摘要
Water is a necessarity to life, and it contains various ions known to be important for biological functions. Cells maintain their vitality through ion exchange and transportation, and it is particularly true for haloarchaea as they possess microbial rhodopsins embedded in cell membrane to serve such need. Among known microbial rhodopsins, halorhodopsin is a light-driven inward chloride pump that maintains osmobalance, while bacteriorhodopsin is a light-driven outward proton pump that subsequently accumulates protons that further drive ATP synthase to produce biological energy, ATP. To study those light-driven ion pumps, electrochemical and other methods are commonly adopted. In first stage of this study, we improved a previously reported ITO-based photocurrent analysis method via investigating its ion selectivity and application in biological molecules. The results showed our modified device to have high proton selectivity and it appeared to have highly linear relation to a wide pH range. Further, signal orientations of transported protons under illumination can be determined. Finally, we found both purified protein, native cells and E. coli cells expressing target proteins can all be applied in this system. The second stage of this study involved in applying this measurement system to resolve a biological system, Haluquadratum walsbyi (H. walsbyi), with two bacteriorhodopsin-like proteins, named HwBR and HwMR. We expressed wild type and mutated proteins in E.coli (C43) to investigate their functional pH change, UV/Vis spectrum, and signals in photocurrent under illumination at both whole cells and purified protein samples. The results showed HwBR is indeed a light-driven outward proton pump with residue D93 in charge of proton releasing, while D104 accerlate proton re-uptake from the cytoplasmic side. HwMR, on the other hand, possesses only weak proton pumping activity. When compared to HwBR, λmax of HwMR was a blue-shift 55 nm in UV/Vis spectrum without significant light-driven proton pumping activity, and possibly non-typical light-driven poroton transportation pathway inside the protein. We therefore proposed it might play signal-modulating role in H. walsbyi. In summary, ITO-based photocurrent method is appropriate for both screening potential light-driven proton-pumping protein and analysis of light-driven proton transportation pathway in any protein.
參考文獻
延伸閱讀
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