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研究生:林修平
研究生(外文):Hsiu-Ping Lin
論文名稱:以嵌合蛋白質探討 HmBRI 第三胞內環之功能
論文名稱(外文):Exploring the Function of the Third Cytoplasmic Loop in HmBRI Using Chimeric Proteins
指導教授:楊啓伸
指導教授(外文):Chii-Shen Yang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:87
中文關鍵詞:嗜鹽性古細菌第三胞內環細菌視紫紅質
外文關鍵詞:Haloarcula marismortuithird cytoplasmic loopbacteriorhodopsin
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嗜鹽性古細菌的視紫紅質可依照其功能被分為四種不同的型態,分別為氫離子幫浦的細菌視紫紅質 (BR)、氯離子幫浦 (HR) 的氯視紫紅質及和正負光趨性相關的感光型視紫紅質 (SRΙ, SRII)。這些視紫紅質皆以 all-trans retinal 做為其發色基團,接受光照後產生異構化變成13-cis retinal 帶動視紫紅質產生結構上的變化而行使生理功能,而後再回到 all-trans retinal 等待下一次的活化。其中細菌視紫紅質、氯視紫紅質和負光趨性感光型視紫紅質的蛋白質結構已被解出,皆為具有七個穿膜α螺旋結構的膜蛋白質,然而這些基本結構相類似的蛋白質,卻具有截然不同的蛋白質特性和生理功能;其中細菌視紫紅質 (BR) 的功能為氫離子幫浦,將胞內的氫離子運送至胞外產生一氫離子之濃度梯度,驅動 ATP 合成酶合成 ATP 給細胞利用。利用結構軟體的分析及過去文獻的報導,我們推測位於第五個α螺旋 (E helix) 和第六個α螺旋 (F helix) 中間的彈性環狀區塊 (E-F loop) 很有可能關鍵性地影響細菌視紫紅質 (BR) 的功能和特性。本研究設計一系列的嵌合蛋白質,將嗜鹽性古細菌 Haloarcula marismortui 中細菌視紫紅質 HmBRΙ 的 E-F loop 置換成阿拉伯芥中 AtGCR1 的 E-F loop,並由大腸桿菌外源表現此一系列嵌合蛋白質,測試其特徵吸收峰及光驅動氫離子幫浦活性,發現這些不同長度的嵌合蛋白質皆保有和 HmBR1 相似的特徵吸收峰,光驅動氫離子幫浦的生理活性也依然存在,說明 HmBRΙ 的 E-F loop 在替換並延長了胺基酸序列後依然保留其原態的功能及維持正常的折疊。然而嵌合蛋白質額外存在有一相異於 HmBRI 之光吸收讀值,顯示 E-F loop 的置換對蛋白質造成某種程度的影響。藉由光週期的量測結果,發現嵌合蛋白質的光週期明顯地在中間態受到延遲,顯示 E-F loop 的置換影響了 HmBRI 自胞內獲取氫離子的過程,也同時證明了 E-F loop 在HmBRI 運送氫離子的機制中,至少扮演部份重要角色。
Chimera WT 是接上阿拉伯芥的 GPCR – AtGCR1 的 HmBRI,過去研究中,AtGCR1 的 E-F loop 是和其下游的 Gα – AtGPA1 進行交互作用的區域。本篇研究嘗試使用 GTP 類似物螢光劑 BODIPYTR-GTP 偵測 Chimera WT 與 AtGCR1 下游的 AtGPA1有無交互作用,以期建立一 GPCRs/Gα 交互作用的研究平台,彌補過去外源表現系統難以表現 GPCRs 的困境。然而,螢光劑易受到緩衝溶液中界面活性劑的干擾,受限於目前無法準確定量界面活性劑,螢光訊號的準確性仍有待評估。若能精確定量界面活性劑,以建構嵌合蛋白質為策略,使用螢光訊號進行 in vitro 的膜蛋白質與可溶蛋白質的交互作用測試,仍具有開發成為一藥物篩選平台的潛力。

Light provides an important energy source for the biosphere on the Earth. Previous studies identified four types of photoreceptors belonging to the rhodopsin family based on different physiological functions in haloarchaea. Bacteriorhodopsin (BR) and halorhodopsin (HR) are known to be light-driven ion transporters while sensory rhodopsins (SRΙ, SRII) were concluded as a phototaxis mediator. These proteins are all the membrane proteins with seven trans-membrane α-helix structure. Even though sharing the similar basic structure, they play different physiological roles in cells. By comparing the features and characteristics of proteins structure, amino acid sequences and the literature reports, we speculate the third cytoplasmic loop (E-F loop) is likely critical in BR’s function. To study of the functional importance of E-F loop in bacteriorhodopsins, a series of chimeric HmBRI from Haloarcula marismortui with the EF loop replaced with various lengths of E-F loop residues from plant AtGCR1 were constructed. The chimeric proteins were over-expressed by Escherichia coli and the purified proteins showed no shift in the maximum absorbance and the proton pump activities were intact. However, the photocycle analysis shows that the chimeric protein delay in the intermediate state for about 2-times slower than HmBRI. Our results suggested the E-F loop significantly affect the characteristics of HmBRI by influencing the efficiency of proton uptake from cytoplasm. The functional importance of E-F loop in bacteriorhodopsin could possibly be established.
Previous study shows that the E-F loop of AtGCR1 interact with its downstream Gα - AtGPA1. In this study, the GTP analogue BODIPYTR-GTP was used as a fluorescent probe to detect the interaction between Chimera WT and AtGPA1. However, the detergent in buffer solution interferes with the fluorescent signal. Currently, there is no suitable method for accurate detergent quantification, so the fidelity of the fluorescence signal has yet to be confirmed. If the detergent can be accurately quantified, the strategy of using chimeric protein and fluorescent probe to monitor the interaction between GPCRs and Gα provides a potential approach for efficient in vitro drug screening.

目錄 i
圖目錄 v
摘要 viii
Abstract x
第一章 緒論 1
第一節 微生物視紫紅質 (Microbial rhodopsin) 1
第二節 細菌視紫紅質 (Bacteriorhodopsin) 3
2.1 HsBR 氫離子運輸之分子機制 5
2.2 HsBR 之光週期 6
第三節 嗜鹽性古細菌 Haloarcula marismortui 9
第五節 E-F loop 相關研究 15
第六節 研究動機與目的 17
第一節 實驗材料與藥品 20
1.1 菌種 20
1.2 質體 20
1.3 藥品 20
第二節 實驗儀器與設備 21
2.1 核酸電泳設備 21
2.2 蛋白質電泳與轉印設備 21
2.3 離心機 22
2.4 氫離子幫浦實驗用儀器 22
2.5光週期測試用儀器 22
2.6 其他 22
第三節 實驗方法 23
3.1 HmBR1-AtGCR1 chimera之重組質體建構 23
3.2 HmBR1-AtGCR1 chimera之突變株質體建構 23
3.3 HmBR1-AtGCR1 chimera 與其系列突變株重組蛋白質之表現及純化 24
3.3.1 蛋白質大量表現 24
3.3.2 破菌與蛋白質粗萃取 25
3.3.3 膜分離與回溶膜蛋白質 25
3.3.4 親和層析法 25
3.4 AtGPA1重組蛋白質之表現及純化 26
3.4.1 蛋白質大量表現 26
3.4.2 破菌與蛋白質粗萃取 26
3.4.3 可溶蛋白質之分離 26
3.4.4 親和層析法 27
3.5蛋白質定性及定量基本分析 27
3.5.1 SDS - PAGE變性膠體電泳 27
3.5.2 西方墨點法及免疫染色分析 28
3.5.3 特徵吸收峰光譜鑑定 28
3.5.4 Bradford蛋白質定量法 28
3.6 氫離子幫浦活性測試 29
3.7 光週期測試 29
3.8 AtGPA1螢光活性測試 30
3.8.1 AlF4- 錯離子測試 30
3.8.2 BODIPYTR-GTP 螢光測試 30
3.8.3 Chimera WT 與 AtGPA1 交互作用測試 31
第三章 結果與討論 32
第一節 建構 HmBRI /AtGCR1 嵌合蛋白質 32
1.1 HmBRI 之 E-F loop 位置預測 32
1.2 AtGCR1 之 E-F loop 位置預測 34
1.3 HmBRI /AtGCR1 之嵌合蛋白質建構 34
第二節 HmBRI /AtGCR1 之嵌合蛋白質大量表現及純化 35
2.1 以Escherichia coli 表現嵌合蛋白質及電泳分析 35
2.2 嵌合蛋白質之光譜分析 38
第三節 Chimera WT 之基本生化特性分析 40
3.1鹽容忍度測試 40
3.2酸鹼容忍度測試 41
第四節 嵌合蛋白質之功能性分析 43
4.1 光驅動氫離子幫浦活性測試 43
4.2 光週期測試 45
4.2.1 Ground state 45
4.2.2 M intermediate state 49
4.2.3 O intermediate state 51
4.2.4 光週期結果總結 53
第五節 嵌合蛋白質與 AtGPA1 交互作用測試 55
5.1以 Escherichia coli 表現 AtGPA1 55
5.2 AtGPA1 於不同鹽濃度及不同 pH 值下之 AlF4- 錯離子活性測試 56
5.3 以螢光劑測試 AtGPA1之活性 58
5.4 以螢光劑測試 Chimera WT 與 AtGPA1 之交互作用 61
第四章 結論 65
第五章 未來展望 69
參考文獻 70
附錄 80



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