並列タイトル等シアノバクテリアに由来する新規ロドプシンの機能解析
一般注記Microbial rhodopsins are widely distributed in many microorganisms and act as light-driven ion pumps, light sensors and light-gated channels. Ion-pumping rhodopsins are classified into three categories: outward proton (H+) and sodium (Na+) pumps and an inward chloride ion (Cl-) pump. After 1999, advances in genome sequencing techniques led to the discovery of novel rhodopsins in various microbes. Recently, I discovered a novel cyanobacterial rhodopsin group that is distinct from other microbial rhodopsins. In part 1 of this thesis, I performed functional characterization of the microbial halorhodopsin (MrHR) from the cyanobacterium Mastigocladopsis repens and its functional conversion to a H+ pump. MrHR has the characteristic residues of a Cl- and H+ pump. Photo-induced ion transport activity measurement revealed that MrHR acts as inward Cl- -pump. The transportable anions are severely restricted to only Cl- and Br-. MrHR bind Cl- near the protonated Schiff base (PSB) to same extent as that of Natronomonas pharaonis halorhodopsin (NpHR). The Cl- transporting photocycle of MrHR is slower than that of other Cl- pumps. Through replacement of the 74th threonine with aspartic acid, MrHR was converted to an outward proton pump. The overall photocycle of T74D resembles that of natural H+ pumps but is prolonged. These results suggest MrHR evolved from a proton pump but not a mature Cl- pump. In part 2 of this thesis, I investigated the photoreactions of MrHR and its mutants. Comparison of the photocycle for transportable (Cl-) and untransportable (I-) anions revealed that the Cl- transfer from the extracellular side to the cytoplasmic side occurs during the L-to-N+O transition. However, the sequence of Cl- release and uptake could not be determined due to the lack of an equilibrium state of the Cl- concentration. The results of Cl- -induced spectral changes showed that R71, T74, S78, and E182 are important for Cl- binding in the dark state. Spectroscopic studies and H+ transfer reaction measurement suggest that Asp-200 protonates during the unphotolyzed state and deprotonates during L-to-N+O transition. The results of ion-transporting measurement suggest the deprotonation of Asp-200 is necessary to transport Cl-. In contrast to this protonation during the dark state and deprotonation during the light state, other Cl- pumping rhodopsins maintain the deprotonation of the corresponding aspartic acid in both the dark and light states. This difference might be attributed to the optimization for Cl- pumping. This study presents the characteristics of a novel chloride-pumping rhodopsin. My findings might be helpful for understanding the functional diversity of microbial rhodopsins and elucidating the mechanism for selecting transportable ions.
(主査) 教授 出村 誠, 教授 金城 政孝, 講師 菊川 峰志
生命科学院(生命科学専攻)
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受理日(W3CDTF)2018-04-03T03:53:09+09:00
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