於大腸桿菌探討微生物視紫質在不同滲透壓環境之光驅動離子傳輸

Probing Light-driven Ion Transportation of Microbial Rhodopsin under Different Osmotic Environments Using E. coli

王赫(Ho Wang)

指導教授 ： 楊啓伸

國立臺灣大學/生命科學院/生化科技學系/碩士(2018年)

https://doi.org/10.6342/NTU201804071

摘要

長久以來，科學界著重研究動物細胞上的離子通道與幫浦，然而真核生物離子通道的結構與功能之成果，大多係建立於原核生物離子通道與幫浦研究基礎上。透過了解微生物離子通道與幫浦的生化與生理性質，將有助於更進一步分子層次上的探討。有別於常用於通道研究的非洲爪蟾 (Xenopus laevis) 卵母細胞，大腸桿菌 (Escherichia coli) 最常被用來表達和研究微生物視紫質 (microbial rhodopsin，MRho) 中一種感光蛋白質—光驅動離子幫浦之離子傳輸功能。但許多因素可能影響細胞膜，進而改變其上離子通道與幫浦之結構與功能；滲透壓便是其中一種。本研究於大腸桿菌表達不同物種的離子型微生物視紫質，並與大腸桿菌細胞本身，進行了於不同滲透壓環境下，全細胞光電流測試 (Whole cell photocurrent measurement)。結果發現，改變外部滲透壓，影響了表達於大腸桿菌上微生物視紫質的傳輸離子活性值。此外，上述之滲透壓改變，對表達不同種類的微生物視紫質 (光驅動離子幫浦) 的大腸桿菌，得到不同影響的活性值。另外透過光學顯微鏡觀察大腸桿菌在不同滲透壓環境下的細胞型態，藉以輔助和分析光電流測試結果之可靠性。本研究以新的角度，探討以大腸桿菌系統研究微生物視紫質的離子傳輸活性和所在環境之影響，未來，更可考慮藉由直接量測以此系統表達之目標蛋白質的離子電導訊號 (ion conductivity) 來了解光驅動離子的傳輸行為。

關鍵字

微生物視紫質 ； 離子通道與幫浦 ； 離子傳輸 ； 滲透壓

並列摘要

Studies of ion channels and pumps have been focused on animal-centric approach in the past, and indeed gained convincing achievements; while the progress of resolving structures and activities of eukaryotic ion channels are still mainly contributed by bacterial experimental models. Other than Xenopus oocytes, Escherichia coli is one of the often-adopted bacterium to express ion pumping microbial rhodopsin. Several factors, including osmotic pressure changes, could influence the physiology and structure of the membrane and further affect performance of expressed ion channels and pumps. This study investigates the ion transportation of ion-type microbial rhodopsins expressed in E. coli, Measuremtents of the light-driven photocurrents of microbial rhodopsins expressed on E. coli membrane under different osmotic environments were conducted. The results show that the external osmolarity affected the ion transportation behaviors of microbial rhodopsins expressed in E. coli. Such osmolarity changes affected differently at different expressed microbial rhodopsins. Observations of the E. coli morphological changes under different osmolarity environments supported the results of photocurrent measurements. We believe such approach provides new insights of using E. coli system to study ion transportation signals of microbial rhodopsins. Also, we propose directly measuring of the ion conductivity on the target protein expressed E. coli cells to investigate ion transportation activity.

並列關鍵字

microbial rhodopsin ； ion channels and pumps ； ion transportation ； osmotic pressure

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