死海嗜鹽方形古菌氫視紫質II上Arg80和Thr199胺基酸對酸耐受度重要性之研究

A Study on the Importance of Residues Arg80 and Thr199 in Conferring Acid Tolerant Proton Pumping of HmBRII

10.6342/NTU201802835

周蔚

細菌氫視紫質 ； 酸耐受度 ； 光電流 ； 氫鍵 ； Bacteriorhodopsin ； Acid-tolerance ； Photocurrent ； Hydrogen Bond

臺灣大學生化科技學系學位論文

2018年

碩士

楊啓伸

繁體中文

本實驗室在2015 年根據細菌氫視紫質 (bacteriorhodopsin) 的序列分析，提出一個新的BR進化枝 (clade)，命名為qR，由Haloarcula marismortui BRII (HmBRII) 和Haloquadratum walsbyi BR (HwBR) 所組成。該研究亦發現qR成員在極酸的環境下仍保有光學穩定性；其中HmBRII則具有已知BR當中，能正常行使氫離子幫浦功能的pH 最低界線；pH 4.0。以模式生物 Halobacterium salinarum 的BR (HsBR) 為例，溶解於pH 5.8以下的環境，會使其無法行使光驅動氫離子幫浦的功能。先前本實驗室由HwBR的蛋白質晶體結構中，發現了由精氨酸82及蘇氨酸201有形成氫鍵，推測此氫鍵能維持位於面向胞外 (extracellular side) 帶正電的beta-loop 結構之穩定性；且此正電特性推測能使其在行使氫離子幫浦功能時，讓原先在蛋白質內部的質子，以協同隔絕方式，抵抗強烈外界氫離子濃度之影響，進而被順利運送到胞外，因而在qR之抗酸性質扮演重要角色。為了進一步證實此氫鍵結構之重要性，本研究使用 HmBRII 作為目標蛋白質，對關鍵氨基酸，即精氨酸80和蘇氨酸199，進行定點突變研究。首先在不同pH 值下測光驅動光電流 (light-driven photocurrent) ，發現突變蛋白質失去在酸性環境下的氫離子幫浦能力。其中，R80E-T199S 雙突變蛋白質功能受挫最顯著，T199S與R80A次之；證據指向T199S 突變的破壞性大。其次，在可見光吸收光譜測量，可發現突變蛋白質特徵吸收峰，對光照後中間態 (M-state) 的比值，較野生型蛋白質低。此顯示突變後影響了蛋白質的整體穩定性，並使其在酸性環境下的特徵吸收峰產生藍移，以T199S 突變蛋白最為顯著，亦顯示胺基酸T199 在此氫鍵系統中的重要性。我們推測T199 相較R80 更重要的可能機制之一，是源由自T199 距BR上保守的質子釋出基團 (proton releasing group) 較近，潛在形成更多原子間作用力，因此影響較為明顯。

Based on phylogenetic analysis of bacteriorhodopsin (BR) sequences, a novel clade of BRs has been proposed by our lab, named qR, consisting of Haloarcula marismortui BRII (HmBRII) and Haloquadratum walsbyi BR (HwBR). It was characterized that both qR members possessed optical stability over a wide range of pH, especially in the acidic region, while HmBRII had a wider functional range toward lower pH (4.0) than any other BR ever observed including Halobacterium salinarum BR (HsBR), and HmBRI. From the crystal structure of HwBR, a unique BC-loop cap structure stabilized by arginine-threonine hydrogen bond was identified. To further explore the important residues or structures in qR, HmBRII was used as the subject to conduct site-directed mutagenesis study of the critical residues, namely, arginine 80 and threonine 199. In this study, ITO-based photocurrent measurements show that mutant proteins have a neutral shifted reversal point, and lose the ability to generate proton pumping signal that the wild type HmBRII possesses at strong acidic pH, R80E-T199S double mutant having the largest impact, followed by T199S and R80A mutant. From UV-Vis spectrum measurements, it was found that mutant proteins are less stable at low pH as a lower ground state absorbance to M-state absorbance ratio was observed along with a larger shift in maximum absorbance. Furthermore, T199S mutant was found to have a larger maximum absorbance blue shift under acidic pH than any other mutant protein. Thus by demonstrating the importance of the R80-T199 hydrogen bond network in the acid resistance of HmBRII protein, we reinforce the significance of the qR grouping and its uniqueness. In addition, the importance of T199 residue is discovered to be more crucial, possibly due to its proximity to the proton releasing group, and other potential interactions.

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