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

• Main Authors: Wei Chou, 周蔚
• Other Authors: 楊啓伸
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/449x4c

碩士 === 國立臺灣大學 === 生化科技學系 === 106 === 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.