感覺型視紫質SRM與其傳導元HtrM影響嗜鹽方形古菌光趨性之分子機制探討

A Study on the Possible Molecular Mechanism of SRM-HtrM Attenuates Phototaxis Responses in Haloarcula marismortui

林育鋒(Yu-Fong Lin)

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(2020年)

https://doi.org/10.6342/NTU202001997

摘要

嗜鹽古細菌利用感覺型視紫質 (SR) 調控光趨性反應。本研究室於2010年發表了嗜鹽方形古菌 (Haloarcula marismortui) 為一株同時具有六種菌式視紫質的嗜鹽古細菌，其中包括三種光驅動離子幫浦和三種SR，除了被廣泛研究的SRI和SRII外，首次發現了SRM以及其配對傳導元HtrM。SRM-HtrM獨特之處在於SRM本身特徵吸收峰波長為503 nm，介於另外兩種感覺型視紫質之間；而HtrM相較於SRI和SRII的傳導元，同樣具有兩個穿膜螺旋，但在胞內側僅具一個HAMP-domain而非兩個，並且缺乏能與下游Che蛋白質作用的methyl-accepting domain。針對SRM-HtrM的特異性，2019年本研究室陳政良同學將SRM-HtrM移植到一株只有SRI和SRII兩種感覺型視紫質的Halobacterium salinarum中，發現活化SRM-HtrM同時會抑制菌體原先對藍光的迴避行為及對紅光的趨向行為，但其中的分子機制尚不清楚。本研究以生物資訊學分析SRM-HtrM發現：（一）HtrM和HtrII的第二個穿膜區進入胞內側的區域有一保守且與功能相關之轉角結構；（二）HtrM的C端有一個可能與Che蛋白質進行交互作用的區域。針對以上兩個功能相關區域進行突變得到突變型SRM-HtrM重組融合蛋白，並完成其特性測試。而為了往後測試HtrM與Che蛋白質間是否有交互作用，從Hm全基因序列中擷取CheW1基因並成功純化出帶有GST-tag之重組蛋白質。另一方面，截至目前為止，SR-Htr的功能性測試皆需要在嗜鹽古細菌中測試菌體光趨性，而嗜鹽古細菌的轉形相當耗時且不易成功。本研究以SRM和SRM-HtrM作為實驗對象，並結合全細胞光電流和蛋白質光電流測試發展出簡易且能快速測試SR與Htr之間是否有適當交互作用之功能性測試。

關鍵字

嗜鹽方形古菌 ； 感覺型視紫質M ； Che蛋白質 ； 光電流

並列摘要

Haloarchaea utilize sensory rhodopsin (SR) to regulate phototaxis responses. Among the haloarchaea, Haloarcula marismortui contains six microbial rhodopsins- three sensory rhodopsin (SR) and three light-driven ion-pump. In addition to SRI and SRII, in 2010, our lab discovered a new kind of SR, SRM, which has a maximum absorbance wavelength of 503 nm, between that of SRI and SRII. When comparing the cognate transducers, HtrM, to HtrI and HtrII, cytoplasmic domain of HtrM possesses only one HAMP-domain rather than two, therefore lacks the methyl-accepting domain to interact with Che proteins. These unique properties raise the questions on both the biological function and possible molecular mechanism of SRM-HtrM. In our past publication, we acknowledge that the blue-light photo-repellent and the red-light photo-attractant responses were both attenuated by light-activation of SRM-HtrM in SRM-HtrM knock-in Halobacterium salinarum cells, however, its molecular mechanism remains unknown. In this study, the bioinformatics analysis results of HtrM show that: (1) HtrM owns a functional-related turn structure behind the second transmembrane helix; (2) the C-terminal of HtrM remains a conserved region that could interact with Che proteins. For further investigation on SRM-HtrM interaction with Che protein, a construction of the HmCheW1 was made. Mutagenesis at those functional-related regions on HtrM and the characterization of those SRM-HtrM mutants were conducted. Until present, the functional assay of SR-Htr can only be done by testing phototaxis responses in haloarchaea cells. Our study combines the whole-cell and protein-based photocurrent assay to develop a more efficient and accurate functional assay for SR-Htr.

並列關鍵字

Haloarcula marismortui ； sensory rhodopsin M ； Che protein ； photocurrent

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