臺灣博碩士論文加值系統

光作為地球上重要的能量來源，其對於生物體的訊息傳遞與生理調控也扮演重要角色。在嗜鹽古細菌 (halophiles) 細胞膜上一般具有四種光感蛋白質，分別是感光型視紫質第一型與第二型 (sensory rhodopsin I,II)，功能為進行訊息傳遞。與細菌視紫紅質 (bacteriorhodopsin, BR)，功能為氫離子幫浦；及氯視紫質 (halorhodopsin, HR)。其中氯視紫質普遍存在於嗜鹽古細菌中，可接收黃光激發而將氯離子往內運輸。藉由在胞內累積氯離子，可使嗜鹽細菌在生長時持續維持滲透壓平衡；此外由於胞內氯離子的增加，造成膜內側負電荷累積，間接增強向內質子動力 (inward proton motive force) 因而使能量的產生更有效率。此外，由於細菌視紫紅質和氯視紫質皆為離子幫浦且兩者在序列上有一定相似性，過去研究曾成功利用突變特定胺基酸 (HsBR 中 D83T與D83S) 而使細菌視紫紅質轉變成一向內傳輸之氯離子幫浦。相反地，若要將氯視紫質變為向外運輸氫離子幫浦，目前所知只能由外加疊氮化物 (azide) 達成。
目前發現的兩種氯視紫質，分別來自Halobacterium salinarum (HsHR) 與Natronomonas pharaonis (NpHR)，其生化特性與光週期已有深入研究，蛋白質結構也都成功解出。本研究新鑑定出兩種氯視紫質，分別來自另兩株嗜鹽古細菌Haloarcula marismortui (HmHR) 和 Haloquadratum walsbyi (HwHR)。將這兩種氯視紫質異源表現純化，可見光光譜掃描顯示其最大特徵吸收峰落在576 nm及573 nm，和NpHR及HsHR非常相近。功能性檢測方面，也以被動氫離子傳輸實驗 (passive proton transport) 證實兩者同為氯離子幫浦。生化特性部分，在氯離子親和性分析實驗及鹼滴定實驗中，所得氯離子解離常數與 pKa 均與前人研究類似。由以上結果整合，相較於其他存在於微生物中之光驅動離子傳輸視紫紅質，氯視紫紅質蛋白質在各方面居有高保守性質，也說明了此類蛋白質對於嗜鹽古生菌的重要性。
另一方面，過去研究顯示沒有任何關於以突變方法，將氯離子幫浦轉換為向外氫離子幫浦的研究；本篇研究嘗試以 HwHR 為例，根據序列比對結果設計三個突變位 (七種突變組合) 將 HwHR 相對應 HsBR 的重要胺基酸依序突變成 HsBR 上所具有的序列；此外，也一同檢驗 D85T 與 D85S 突變是否能在 Haloarcula marismortui 細菌視紫紅質中得到相同效應。經過蛋白質表現、純化及生理測試，HmBRI D83S 突變蛋白質具有被動氫離子傳輸活性，HmBRI D83T 突變蛋白質則轉換為向內氫離子傳輸蛋白質。然而，所有HwHR突變蛋白質皆未觀察到功能上的變化，甚至數個突變蛋白質皆成為 apoprotein 或在異源表現上遭遇困難。造成此現象的原因可能為引入之突變胺基酸使視黃醛分子無法順利與蛋白質結合，也顯示了細菌視紫紅質在演化過程中應早於氯視紫質，因而這些重要胺基酸的改變於功能上無法逆轉。

Light serves as a crucial environmental signal to all organisms on the Earth and closely involves in physiological signaling and regulations. For halophiles widely found in NaCl-saturated ponds, most of them encode archaeal rhodopsins to harvest different wavelengths of light for either ion transportation or as sensory mediator. One of these rhodopsins, halorhodopsin (HR), was found to be an inward light-driven chloride ion transporter which ubiquitously exists in halophilic archaea. HR contains retinal as chromophore and utilizes 576 nm of light to transport chloride and other halides into cytoplasm so as to maintain osmotic balance during cell growth. By cooperating with light-driven proton transporter bacteriorhodopsin, HR generates a positive outside membrane potential, therefore enhancing the inward-directed proton motive force.
Since the similarity between two ion pumps, HR and BR, previous studies have investigated the possibility to convert BR to HR or vice versa. So far, preliminary results indicated that conversion of BR to HR can be accomplished via introducing D83T or D83S mutations, and HR possessed a BR-like photocycle in the presence of azide. However, no published studies have reported the conversion of HR to BR by using point mutagenesis.
HR isolated from Halobacterium salinarum (HsHR) and Natronomonas pharaonis (NpHR) were well-investigated. In this study, we reveal two new HRs, HmHR (from Haloarcula marismortui) and HwHR (from Haloquadratum walsbyi), both are functionally overexpressed and purified from E.coli C43 (DE3). The absorption maximum of HmHR and HwHR locates at 576 nm and 573 nm, respectively, which is really close to known wavelength (576 nm). Upon green laser illumination, both of them exhibit passive proton uptake activity. Furthermore, spectral experiments of binding affinity and pH replacement assay also display certain similarities with HsHR and NpHR. These results lead to the conclusion that HRs in haloarchaea share more conserved properties rather than other ion translocating microbial rhodopsins, which suggests its physiological significance through evolution.
On the other hand, the interconversion between BR and HR in rhodopsin systems of Haloarcula marismortui or Haloquadratum walsbyi is also examined in this study. The results showed that D83S mutation in HmBRI seemed to successfully convert BR to an inward chloride pump. Another mutant of HmBRI, D83T, was likely to alter HmBRI into an inward proton pump. On the other hand, all HwHR mutants designed to convert HR to BR were failed probably because of the obstruction of retinal uptake process. It is possible that conversion of HR to BR cannot be accomplished by alignment-based mutagenesis; the other explanation is that BR was earlier in evolution than HR, therefore substitution of Asp by Thr or Ala was irreversible in functions.

Contents i
List of tables iii
List of figures v
Abstract viii
中文摘要 x
Chapter One: Introduction 1
1.1 Archaeal rhodopsins 1
1.2 The Ion-translocating microbial rhodopsin family 4
1.3 Halorhodopsin (HR) 6
1.3.1 Structures 6
1.3.2 Photocycle 8
1.3.3 Transportation mechanism 10
1.3.4 Physiological function 12
1.3.5 Mutagenesis studies 14
1.3.6 Interconversions of BR and HR 16
1.3.7 Applications 17
1.4 Overall features of Haloarcula marismortui and Haloquadratum walsbyi 20
1.4.1 Haloarcula marismortui 20
1.4.2 Haloquadratum walsbyi 21
1.5 Rhodopsins in Haloarcula marismortui and Haloquadratum walsbyi 22
1.6 Purpose of this study 23
Chapter Two: Material and methods 25
2.1 Bioinformatics analysis 25
2.2 Bacterial strains 25
2.3 Construction of Expression Plasmids 26
2.3.1 Construction of Expression Plasmids of HR with 6xHis-Tag 26
2.3.2 Construction of BR and HR mutants 26
2.4 Protein overexpression and purification of photoreceptors of Haloarcula marismortui / Haloquadratum walsbyi 28
2.4.1 Traditional purification method 28
2.4.2 Heat treatment purification 29
2.5 SDS-PAGE and western blotting 30
2.6 UV/Vis spectrum scanning 31
2.7 pKa determination 31
2.8 Binding affinity assay 32
2.9 Ion transporting activity assay 33
2.9.1 Ion transporting activity measurement 33
2.9.2 Ion transporting activity measurement with CCCP 34
2.10 Photocycle measurements 35
Chapter Three: Investigation of HmHR and HwHR 37
3.1 Sequence analysis of HRs 37
3.2 Expression and purification of HRs 42
3.3 Functional analysis of HRs 44
3.4 pH-dependent spectral changes of HRs 47
3.5 Binding affinity to chloride of HRs 50
3.6 Laser flash-induced transient absorption changes of HRs 53
3.7 Conclusions 55
3.8 Discussions 58
Chapter Four: Interconversions of BR and HR 61
4.1 Sequence alignment of BRs and HRs 61
4.2 Overexpression and purification of mutants 63
4.3 Functional analysis of BR and HR mutants 67
4.4 Conclusions 69
4.5 Discussions 70
Future Perspectives 72
References 73

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