| Summary: | 碩士 === 國立臺灣大學 === 生化科技學系 === 103 === 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.
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