鹽方扁平古菌中兩種被預測為細菌視紫紅質之特性研究

Abstract

微生物視紫紅質自 1973 年開始，在模式生物嗜鹽古細菌Halobacterium salinarum 發現四種利用光源為能量行使生理功能的視紫紅質，其中分為調節離子濃度的離子幫浦型與感應光趨性的感光受器型，細菌視紫紅質即為前者，其為最早且研究最為透徹的古生菌視紫紅質，利用光源為能量將氫離子送出胞外，造成氫離子濃度梯度，進而帶動三磷酸腺苷生成系統產生能量。 古生菌 Haloquadratum walsbyi 在 2006 年基因體解碼資料中，有三種預測會表達視紫紅質的基因，包括兩種類細菌視紫紅質及一種類感光型視紫紅質。其中視紫紅質基因bop1 和 bop2 皆被預測為類細菌視紫紅質，其轉譯後蛋白被分別命名為HwBR 和 MR( middle rhodopsin )。本研究旨在研究上述兩類細菌視紫紅質間之特性，並探討為何 H. walsbyi 內擁有兩細菌視紫紅質系統。經序列比對與演化樹比對，HwBR 和 MR有很高的相似度，且基因經大腸桿菌表達系統表達純化後，兩者之特徵吸收峰分別為552 奈米和 488奈米。HwBR 經氫離子幫浦測試後，擁有如細菌視紫紅質般的能力，惟 MR 即使經其特徵吸收峰波長 470奈米雷射光源刺激亦無明顯反應。在光週期測試方面，HwBR 光週期如細菌視紫紅質般，約為200毫秒，然而MR光週期達2秒，類似感光型視紫紅質。進一步利用點突變研究，分別對兩基因突變兩處細菌視紫紅質保守且重要之序列，其影響結果HwBR 與細菌視紫紅質相同，MR則非。爰此，整體研究可以清楚推論H. walsbyi與H. marismortui之兩細菌視紫紅質系統相異，前者只包含一種細菌視紫紅質和一種稱為MR之獨特視紫紅質，其被獨立分類於離子型與感光型間，其生理功能尚需研究與探討，而Asp96之保守性也不再是判斷為細菌或感光型視紫紅質之依據。

Studies in microbial rhodopsins have been focused on those found in Halobacterium salinarum since 1973. In the past, the studies in halophilic archaea identified four kinds of retinal-binding proteins and they use light as energy source to mediate different physiological functions. Among them, two main functions are identified: light-driven ion transporters and sensory receptor for phototaxis responses. Bacteriorhodopsin belong to ion-transporter type and is the first and best-understood archaeal rhodopsin which can pump protons out of cell upon light illumination to induce a proton gradient, which further lead to ATP synthesis via F1Fo ATP synthase system. Haloquadratum walsbyi genome was completed in 2006 and a total of three retinal-binding proteins were predicted, including two bacteriorhodopsins-like and one halorhodopsin-like chloride pumping photoreceptor. The two opsin genes in H. walsbyi, bop1 and bop2, were those that identified and assumed to encode two bacteriorhodopsin-like proteins, and their protein products are named HwBR and MR, respectively. The goal of this study is to compare the features between HwBR and MR to further understand whether H. walsbyi indeed posses a two-bacretiorhodopsin system. The protein sequence alignment and phylogenetic tree analysis showed the high identity of HwBR and MR. The genes HwbopI and HwbopII were cloned and expressed in E. Coli C43(DE3) for biochemical property studies, and the maximum absorbance were 552 nm and 488 nm, respectively. The light-driven proton pumping activity showed that HwBR to have light-driven proton transportation function as that found in bacteriorhodopsin, while MR showed no such activity at all, even when being activated with the 470nm laser beam. The photocycle kinetic measurements showed HwBR to have a 200 msec photocycle time just like bacteriorhodopsin, while it was a 2-second for MR, a time course similar to those found in sensory rhodopsins. Further different mutagenesis studies at Asp85 and Asp96, two conserved and functionally critical residues found in bacteriorhodopsin, found only HwBR showed¬- the same impact in photocycle kinetics as those reported in HsBR but not in MR. The overall results in this study generated three conclusions: i) H. walsbyi does not contain a two-bacteriorhodopsin system as that identified in Haloarcula marismortui ; it has one bacteriorhodopsin-like protein and one unique rhodopsin, MR, ii) MR possesses biochemical properties that can only be classified as one that between ion-type and sensory-type. Further experiments are needed to perform to determine the function of MR. iii) The existence of a corresponding resiude Asp96 as in HsBR no longer clearly separates a bacteriorhodopsin-type retinal-binding protein from a sensory rhodopsin-type.