表現及測定離子運輸型微生物視紫質之大腸桿菌原生包膜體系統

Overexpression and functional probing of ion-type microbial rhodopsins in an Escherichia coli derived spheroplast system

林國禎(Guo Zhen Lim)

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(2022年)

https://doi.org/10.6342/NTU202201528

摘要

源自嗜鹽古生菌的感光膜蛋白質——視紫質蛋白質因其高時間及空間解析度的光控性，常被大量表現於大腸桿菌中以利研究及大量純化。但這些視紫質對細胞生理的影響及其潛在的生理意義卻還未有完善的研究。經過多年的探索，不同功能的微生物視紫質蛋白質（microbial rhodopsin）在古生菌、細菌及低等真核生物中陸續被發現。目前主要的分類方法將已知功能的視紫質蛋白質分爲離子運輸型（ion translocation type）與感受型（sensory type）視紫質兩大類。其中，源自Natronomonas pharaonis嗜鹽古生菌的氯視紫質蛋白質（halorhodopsin，NpHR）是一種可被光控的氯離子幫浦，在受到光照後能以毫秒等級的速率將氯離子主動運輸到胞內。因此halorhodopsin在近期常被應用於光控基因生物學（optogenetics），作爲神經抑制型的感光蛋白質工具。然而，其對於神經細胞過極化效果過於強烈而導致細胞生理機制受影響。先前的研究也發現，NpHR除了擁有一般氯視紫質的氯離子幫浦功能外，也意外的可偵測到氫離子胞內循環的訊號；在目前已知的物種中，只有Haloquadratum walsbyi halorhodopsin（HwHR）擁有類似於NpHR的功能特徵。爲深入研究NpHR及其突變株對於細胞生理的影響，本實驗利用大腸桿菌去除細胞壁之原生包膜體（spheroplast）作爲可快速表現及測試仿原生膜上視紫質的系統。在確認視紫質蛋白成功表現於原膜體之細胞膜後觀察大腸桿菌表現視紫質蛋時的生長曲線。接著，進行原膜體光電流實驗確認蛋白質功能，並透過顯微影像觀察照光後視紫質原膜體的形態改變和計數統計。本研究成功建構利用此大腸桿菌原膜體作爲模式測試平臺，觀察離子運輸型蛋白質在細胞膜上之功能改變，並推測NpHR對於細胞膜的損害是因急速的氯離子幫補功能而導致滲透壓超出細胞膜可能承受的範圍所引起。本研究也發現，當Trp127被突變爲Phe時，較野生型緩慢且只保有氯離子幫浦功能的NpHR突變株對於細胞的傷害有顯著降低。因此，NpHR-W127F有機會成爲比野生型更適合作爲光控基因生物學應用的感光蛋白質工具。

關鍵字

微生物視紫質 ； 氯視紫質 ； 法老嗜鹽鹼單胞菌 ； 大腸桿菌 ； 原膜體 ； 光控基因生物學

並列摘要

The light-sensitive seven-transmembrane proteins originated from haloarchaea, microbial rhodopsins (mRhos), are widely cloned in the Escherichia coli system for functional and atomic structural studies due to their light controllability with exceptional spatial and temporal resolutions. However, the inherent physiological significance of these light-driven bio-machines and their effects on cell physiology is yet to be fully understood. Years of discoveries have collected an arsenal of mRhos from archaea, eubacteria, and lower eukaryotes, each bearing different functionalities. The current paradigm has classified known mRhos into two main categories according to their reported functions, namely the ion translocation type and sensory type mRhos. Among them, halorhodopsin from Natronomonas pharaonis (NpHR) is a light-driven chloride pump capable of rapid chloride transportation from the extracellular environment to the cytoplasm in milliseconds. NpHR is therefore frequently utilized in recent optogenetic applications as a photoreceptor tool to realize light-controlled neuronal inhibition. Nevertheless, strong hyperpolarization induced by NpHR on neurons often leads to physiological complications in the target cells. Previous studies reported that apart from the chloride pumping function typical to halorhodopsins, NpHR possesses a unique proton circulation signal; Haloquadratum walsbyi halorhodopsin (HwHR) is the only other HR known to own this property. To investigate the physiological effects of NpHR and its variants on cells, this study exploited cell-wall-deficient E. coli spheroplasts to develop an efficient expression platform to mimic a cell-based system. We conducted several experiments for that purpose, including: 1) confirmation of mRho localization on spheroplast membranes; 2) growth curve monitoring of E. coli expressing mRho; 3) the functional probe of mRhos with spectrophotometry, photocurrent and photocycle assays; 4) microscopy imaging to observe cell morphological changes; 5) and cell viability assay. By employing the spheroplast platform, we reproduced the negative impact NpHR has on host physiology. We proposed that the damage by NpHR arose from its rapid photocycle kinetics and aggressive chloride pumping activity, causing the cells to experience a drastic change in osmotic pressure beyond their normal homeostasis range. Moreover, we showed that a mutation of Trp127 residues to Phe on NpHR eliminated its unique proton signal and slowed the photocycle kinetics. However, NpHR-W127F retained its chloride pumping activity and reduced the damaging effect of NpHR on spheroplasts. Thus, we propose that NpHR-W127F might be more suited for optogenetics applications as an optogenetic photoreceptor tool.

並列關鍵字

microbial rhodopsin ； halorhodopsin ； Natronomonas pharaonis ； Escherichia coli ； spheroplast ； optogenetics

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