鹽方扁平古菌上氯視紫蛋白質光驅動離子傳遞能力之探討

Investigation on light-driven ion translocation capability of halorhodopsin from Haloquadratum walsbyi

陳筱儒(Xiao-Ru Chen)

指導教授 ： 楊啟伸

國立臺灣大學/生命科學院/生化科技學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02399

摘要

嗜鹽古細菌 (haloarchaea) 生存在高鹽的嚴苛環境中，菌體中存在許多蛋白質協助抵抗極端環境，其中一類為微生物視紫蛋白質(Microbial rhodopsin)，當受到光刺激後會有不同的功能，主要可分為兩大類，光驅動離子幫浦及光感受器。在光驅動離子幫浦中，其中一種為氯視紫蛋白質 (halorhodopsin, HR)，目前已知是一種光驅動氯離子幫浦，可以維持古生菌的細胞滲透壓。當受光刺激後，氯離子會由胞外送至胞內，氫離子被動運輸至細胞內。在本研究中，透過對於氫離子敏感的光電流測試，進一步測量光驅動氫離子釋出的活性。結果發現當 Halobacterium salinarum (Hs) 及 Haloarcula marismortui (Hm) 上的氯視紫蛋白質 (HR) 以 E.coli 表現時，質子的確被動進入膜內。但是若將蛋白質純化後，以蛋白質溶液進行光電流測試，照光前後並不會產生質子梯度。然而，由結果發現在 Haloquadratum walsbyi 中的氯視紫蛋白質 (HwHR) 有別於 HsHR 及 HmHR，無論是以表現於 E.coli 的菌液或是純化後的蛋白質溶液，進行光電流測試，皆有質子梯度的產生。為了找出在 HwHR 中，與細菌視紫蛋白質 (bacteriorhodopsin) 相似的質子幫浦活性所涉及的胺基酸，找出帶負電胺基酸進行點突變後，發現 D254N 質子釋出的活性消失。D254N-HwHR在特徵吸收波長上往短波長移動 15 nm，光週期則比 wild type 慢 6 倍。綜合上述結果， Asp254 對於光驅動質子釋出及視黃醛重新異構化的效率上扮演重要角色。

關鍵字

嗜鹽古細菌 ； 微生物視紫蛋白質 ； 氯視紫蛋白質 ； 光週期

並列摘要

Microbial rhodopsins response to light and function as light-driven ion transportation or light sensor. Halorhodopsin (HR), one of these microbial rhodopsins, is known to be a light-driven inward chloride pump for osmolarity maintenance at least in haloarchaea. During inward transportation, chloride ions are proposed to passively carry protons across the membrane. In this study, we used a proton sensitive assay, photocurrent measurement, to measure light-driven proton releasing activity. Here we found halorhodopsin in Halobacterium salinarum (HsHR) and Haloarcula marismortui (HmHR), the protons indeed passively transported across the membrane when they were expressed in E. coli cells, but in the purified protein level, no light-driven photocurrent was recorded, indicating no proton gradients were formed inside and outside protein upon illumination. However, halorhodopsin in Haloquadratum walsbyi (HwHR) generated positive in both cell-based and protein photocurrent measurement. In order to find the residue(s) in HwHR mediating such bacteriorhodopsin-like proton activity, we mutated several negative residues and found D254N eliminate such activity in both cell-based and protein experiments. D254N-HwHR underwent a 15-nm blue-shifted in maximum absorbance and the recovery time of photocycle was ~6 time slower when compared to wild type. According to these results, we conclude Asp254 as the critical residue for light-driven proton releasing and the efficiency of retinal reisomerlization.

並列關鍵字

haloarchaea ； microbial rhodopsin ； halorhodopsin ； photocycle

參考文獻

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