| Summary: | 碩士 === 臺灣大學 === 微生物與生化學研究所 === 98 === Light serves as a crucial environmental signal to all organisms on the Earth and closely involves in physiological signaling and regulations. For halophiles widely found in NaCl-saturated ponds, most of them encode archaeal rhodopsins to harvest different wavelengths of light for either ion transportation or as sensory mediator. One of these rhodopsins, halorhodopsin (HR), was found to be an inward light-driven chloride ion transporter which ubiquitously exists in halophilic archaea. HR contains retinal as chromophore and utilizes 576 nm of light to transport chloride and other halides into cytoplasm so as to maintain osmotic balance during cell growth. By cooperating with light-driven proton transporter bacteriorhodopsin, HR generates a positive outside membrane potential, therefore enhancing the inward-directed proton motive force.
Since the similarity between two ion pumps, HR and BR, previous studies have investigated the possibility to convert BR to HR or vice versa. So far, preliminary results indicated that conversion of BR to HR can be accomplished via introducing D83T or D83S mutations, and HR possessed a BR-like photocycle in the presence of azide. However, no published studies have reported the conversion of HR to BR by using point mutagenesis.
HR isolated from Halobacterium salinarum (HsHR) and Natronomonas pharaonis (NpHR) were well-investigated. In this study, we reveal two new HRs, HmHR (from Haloarcula marismortui) and HwHR (from Haloquadratum walsbyi), both are functionally overexpressed and purified from E.coli C43 (DE3). The absorption maximum of HmHR and HwHR locates at 576 nm and 573 nm, respectively, which is really close to known wavelength (576 nm). Upon green laser illumination, both of them exhibit passive proton uptake activity. Furthermore, spectral experiments of binding affinity and pH replacement assay also display certain similarities with HsHR and NpHR. These results lead to the conclusion that HRs in haloarchaea share more conserved properties rather than other ion translocating microbial rhodopsins, which suggests its physiological significance through evolution.
On the other hand, the interconversion between BR and HR in rhodopsin systems of Haloarcula marismortui or Haloquadratum walsbyi is also examined in this study. The results showed that D83S mutation in HmBRI seemed to successfully convert BR to an inward chloride pump. Another mutant of HmBRI, D83T, was likely to alter HmBRI into an inward proton pump. On the other hand, all HwHR mutants designed to convert HR to BR were failed probably because of the obstruction of retinal uptake process. It is possible that conversion of HR to BR cannot be accomplished by alignment-based mutagenesis; the other explanation is that BR was earlier in evolution than HR, therefore substitution of Asp by Thr or Ala was irreversible in functions.
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