臺灣博碩士論文加值系統

氯視紫紅質 (halorhodopsin, HR) 是存在於古細菌 (haloarchaea) 之中的一種七穿膜區且含視黃醛蛋白質，目前已知是一種光驅動氯離子幫浦。然而過去有研究發現，不同物種間的氯視紫紅質不完全相同，在結構和傳遞機制上存在著些微的差異。在此研究中我們使用氧化铟锡 (ITO, indium-tin oxide) 材料來測試由光驅動氫離子釋出所造成的光電流 (photocurrent) 產生之情形，發現了NpHR (halorhodopsin from Natronomonas pharaonis) 產生獨特的電流訊號，的確指出不同物種氯視紫紅質之間的不同。針對若干高度保留且和氯離子穩定相關的胺基酸進行點突變，發現特定帶電的胺基酸一旦突變後光電流訊號即消失。再者，針對光電流產生和光週期相對應的時間設計了一個可以同時測量光電流和光週期的系統。最後我們提出了三種可能的傳遞機制模型，並試圖解釋這個氯視紫紅質之間的差異。總而言之，此研究提供了一個高解析度的方法來探討氯離子-協助之氫離子傳輸的機制。

Halorhodopsin (HR) is a seven-transmembrane (7TM) retinylidene protein from haloarchaea that is commonly known to function as a light-driven inward chloride pump. However, previous studies have indicated that despite the general characteristics that most HRs share, HRs from distinct species differ in many aspects. We present indium-tin oxide (ITO)-based photocurrent measurements that demonstrate light-induced signal generated by proton release, which is observed solely in NpHR through purified protein-based assays, indeed showed HRs are not all identical. Mutagenesis studies were conducted on several conserved residues considered critical for chloride stability in HRs. Intriguingly, the photocurrent signals were eliminated after specific point mutation. Furthermore, the photocurrent and various photocycle intermediates were recorded simultaneously. We summarized three possible mechanisms in attempt to explain the differences among HRs. Overall, this approach provides a high-resolution method for further investigation of the chloride-associated proton-translocation mechanism.

Abstract 7
中文摘要 8
Chapter One: Introduction 9
1.1 Microbial rhodopsin 9
1.2 Halorhodopsin (HR) 12
1.3 Structure of halorhodopsin 14
1.4 Photocycle of halorhodopsin 16
1.5 Transportation mechanism 17
1.6 Functional assays 19
1.7 Comparison of BR and HR 21
1.8 Natronomonas pharaonis 22
1.9 Purpose of this study 24
Chapter Two: Materials and Methods 26
2.1 Bacterial Strain 26
2.2 Plasmid Construction 26
2.3 List of chemical drugs 27
2.4 Instrument and equipment 28
2.5 Experimental methods 30
Chapter Three: Results and Discussions 40
3.1 Indium tin oxide (ITO)-based study 40
3.2 Function assay for HRs via pH change measurement 44
3.3 Illumination-driven photocurrent measurements for HRs 45
3.4 Finding the origin of signal via Cl--free-/ MES-/ pH-dependent –photocurrent 47
3.5 Mutagenesis study 50
3.6 Photocycle measurement 56
3.7 pH-dependent maximum absorbance 59
3.8 Simultaneous measurement of photocycle and photocurrent 61
Chapter Four: Summary 63
Chapter Five: Future Perspectives 67
References 68

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