Functional analysis of a novel cyanobacterial rhodopsin

• Material Type: 博士論文
• Title: Functional analysis of a novel cyanobacterial rhodopsin
• Author/Editor: 長谷見, 崇俊
• Author Heading: 長谷見, 崇俊
• Publication, Distribution, etc.: Hokkaido University
• Publication Date: 2017-03-23
• Publication Date (W3CDTF): 2017-03-23
• Alternative Title: 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
• Contributor: 出村, 誠; 金城, 政孝; 菊川, 峰志
• Degree grantor/type: 北海道大学
• Date Granted: 2017-03-23
• Date Granted (W3CDTF): 2017-03-23
• Dissertation Number: 甲第12725号
• Degree Type: 博士(生命科学)
• Conferring No. (Dissertation): 甲第12725号
• Text Language Code: eng
• NDC: 400
• Target Audience: 一般
• Note (General): 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）
• DOI: 10.14943/doctoral.k12725; https://doi.org/10.14943/doctoral.k12725
• Persistent ID (NDL): info:ndljp/pid/11063811; https://dl.ndl.go.jp/pid/11063811
• Collection: 障害者向け資料
• Collection (Materials For Handicapped People:1): テキストデータ
• Collection (particular): 国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文; https://dl.ndl.go.jp/collections/A00014
• Acquisition Basis: 博士論文（自動収集）
• Date Accepted (W3CDTF): 2018-04-03T03:53:09+09:00
• Date Created (W3CDTF): 2017-03
• Format (IMT): PDF
• Access Restrictions: 国立国会図書館内限定公開
• Service for the Digitized Contents Transmission Service: 図書館・個人送信対象外
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• Periodical Title (URI): http://dx.doi.org/10.14943/doctoral.k12725; http://hdl.handle.net/2115/68558
• Is Referenced By (URI): http://hdl.handle.net/2115/65558
• Data Provider (Database): 国立国会図書館 : 国立国会図書館デジタルコレクション; https://dl.ndl.go.jp