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標題: | 利用HEBR的光驅動幫浦活性偵測TaHeR的方向性 Using the light-driven pump activity of HEBR to detect the orientation of TaHeR |
作者: | Wei-Lin Chen 陳韋霖 |
指導教授: | 楊啓伸(Chii-Shen Yang) 楊啓伸(Chii-Shen Yang | csy.ntu@gmail.com | ), |
關鍵字: | Heliorhodopsins,HEBR,光電流,光週期,N端, Heliorhodopsins,HEBR,photocurrent,photocycle,N ternimus, |
出版年 : | 2022 |
學位: | 碩士 |
摘要: | Heliorhodopsins(HeRs)是一個能被大約540奈米光波長所激發的微生物視紫質家族,是用總體基因學才發現的。Heliorhodopsins和其他微生物視紫質相比有兩個不同之處1.HeRs的N端被人提出位於胞內而非在細胞外,和目前所有已知的七穿膜蛋白質不同2.HeRs僅在革蘭氏陽性菌和單層微生物中發現。與其他視紫紅質相比HeRs沒有離子幫浦的活性,光週期也非常緩慢。本研究的主要目的是檢查細胞中HeRs的N端可能的方向。我們選定TaHeR(T. archaeaon heliorhodopsin)研究,將HeR與一種表達標籤Haloarcula marismortui Bacteriorhodopsin I D94N 突變體HEBR(HmBRI-D94N)融合,該突變體已知具有光驅動的向外質子幫浦送活性。我們將HeR的N端分別設計為在胞外的HEBR-HtrI-HeR,和在胞內的HEBR-HeR。HEBR-HeR和HEBR-HtrI-HeR分別被表現在大腸桿菌後並純化,HEBR-HeR與HEBR顏色較為相近。在紫外光-可見光光譜圖下,只有HEBR-HeR有明顯的特徵吸收峰。在細胞濃度相同的全細胞光電流下顯示HEBR-HeR在溶液中的光驅質子幫浦信號強於HEBR-HtrI-HeR。在蛋白質濃度相似的蛋白質光電流則顯示HEBR-HeR和HEBR-HtrI-HeR在溶液中的光驅質子幫浦信號強度是相同的。而且,將HEBR-HeR和HEBR-HtrI-HeR中HEBR突變成HEBRD83N的無光驅動氫離子幫浦活性突變體,全細胞光電流則消失,顯示所測得之光電流來自HEBR,而HeR沒有光驅動幫浦活性,並且HeR的N端在胞內,顯然不影響HEBR功能。在光週期測量下,也顯示只有HeR的N端在胞內的HEBR-HeR保留了HeR的O態光週期特性。綜合上述結果,HEBR在HEBR-HeR中似乎具有不受影響的特性,並且 HEBR-HeR中的HeR比HEBR-HtrI-HeR中更好地保留了其生化和生物物理特性。最後,我們並進一步分析了HEBR和HeR中表面殘基的電荷特性,發現HeR的N端位於胞內的HEBR-HeR融合蛋白質中,表面電荷在胞內和胞外的趨勢是一致的。因此,我們得出結論,當HeR的N端位於胞內時,的確更為穩定。 Heliorhodopsins (HeRs) are a family of microbial rhodopsins that are excited by light wavelengths around 540 nanometers and were discovered using metagenomics. Compared with other microbial rhodopsins, heliorhodopsins have two differences: 1. The N-terminus of HeRs has been proposed to be located inside the cell rather than outside the cell, which is different from all the known seven transmembrane proteins. 2. It is found in Gram-positive bacteria and monoderm. Compared with other rhodopsins, HeRs have no ion pump activity and a very slow photocycle. The main purpose of this study was to examine the possible orientation of the N-terminus of HeRs in cells. We selected TaHeR (Thermoplasmatales archaeaon heliorhodopsin) for research, and fused HeR with an expression tag Haloarcula marismortui Bacteriorhodopsin I D94N mutant HEBR (HmBRI-D94N), which is known to have light-driven outward proton pumping activity. We designed the N-terminus of HeR as HEBR-HtrI-HeR in extracellular and HEBR-HeR in intracellular, respectively. HEBR-HeR and HEBR-HtrI-HeR were expressed in E. coli and purified, respectively. HEBR-HeR and HEBR were similar in color. Under the UV-Vis spectrum, only HEBR-HeR has obvious characteristic absorption peaks. The light-driven proton pump signal of HEBR-HeR in solution was stronger than that of HEBR-HtrI-HeR under the whole-cell photocurrent at the same cell concentration. Protein photocurrents at similar protein concentrations showed that the light-driven proton pump signal intensities of HEBR-HeR and HEBR-HtrI-HeR in solution were the same. Furthermore, when HEBR in HEBR-HeR and HEBR-HtrI-HeR was mutated to a no light-driven ion-pumping mutant of HEBRD83N, no signal appeared in the whole-cell photocurrent, indicating that the measured photocurrent was from HEBR, while HeR had no light-driven ion pump activity. When the N-terminus of HeR is intracellular, it does not affect HEBR function. Under photocycle measurement, shown that HEBR-HeR with only N-terminus of HeR in the cell retains the O-state of HeR. Taken together the above results, HEBR appears to have unaffected properties in HEBR-HeR, and HeR in HEBR-HeR retains its biochemical and biophysical properties better than in HEBR-HtrI-HeR. Finally, we further analyzed the surface charge characteristics in HEBR and HeR, and found that the trend of surface charge in the intracellular and extracellular was consistent in the HEBR-HeR fusion protein which N-terminus of HeR was located in the intracellular. Therefore, we conclude that HeR is indeed more stable when its N-terminus is intracellular. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86062 |
DOI: | 10.6342/NTU202203342 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2022-09-16 |
顯示於系所單位: | 生化科技學系 |
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U0001-1309202213062100.pdf | 4.28 MB | Adobe PDF | 檢視/開啟 |
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