{"created":"2023-06-19T10:29:53.277991+00:00","id":9955,"links":{},"metadata":{"_buckets":{"deposit":"e5d189e3-ec14-4a50-9a96-dcc4becc2526"},"_deposit":{"created_by":18,"id":"9955","owners":[18],"pid":{"revision_id":0,"type":"depid","value":"9955"},"status":"published"},"_oai":{"id":"oai:muroran-it.repo.nii.ac.jp:00009955","sets":["41:227"]},"author_link":["56152"],"item_81_date_granted_17":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2019-03-25"}]},"item_81_degree_grantor_10":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_language":"ja","subitem_degreegrantor_name":"室蘭工業大学"},{"subitem_degreegrantor_language":"en","subitem_degreegrantor_name":"Muroran Institute of Technology"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"10103","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_81_degree_name_11":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（工学）","subitem_degreename_language":"ja"}]},"item_81_description_25":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_81_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"Microbial rhodopsins are typical membrane proteins with seven transmembrane alpha-helices (helices A-G). This type of protein has a retinal molecule as the chromophore. The microbial rhodopsins use the retinal chromophore attached at a conserved lysine residue to absorb photons for light-energy conversion and light-signal transduction. The molecular mechanisms of microbial rhodopsins have been discussed at the individual amino acid level. In this study, we have prepared amino acid replaced mutants for sensory rhodopsin II from Halobacterium salinarum (HsSRII), and archaerhodopsin discovered by our group from salt lake in Inner Mongolia of China (HeAR). Their photochemical properties were studied by flash photolysis and the the function of amino acids which have been not reported until now was clarified. HsSRII is a negative phototaxis receptor, which makes bacterium avoid blue-green light. In this study. The amino acid replaced mutants of aspartic acid 103 (D103) of HsSRII were prepared and analyzed with flash photolysis. Aspartic acid 103 of HsSRII corresponds to D115 of bacteriorhodopsin (BR). This amino acid residue D115 is functionally important in BR. Aspartic acid D103 were replaced with asparagine (D103N) or glutamic acid (D103E). The work revealed that a substitution of D103 with asparagine (D103N) or glutamic acid (D103E) can resulted in large changes in HsSRII photocycle. These alterations include acceleration of the decay of the M intermediate, prolonging the lifetime of intermediates in the later part of photocycle, and appearance of a long-lived shorter-wavelength photoproduct. Thus, D103 of HsSRII may play an important role in regulation of the photocycle of HsSRII. HeAR is a light driven outward proton pump like to BR. All key functional amino acid residues for proton pump of BR are conserved in HeAR. In the present work, we payed attention to amino acids which are only conserved in ARs, prepared the mutants T164A, S165A and T164A/S165A (T164 and/or S165 were replaced with an alanine) and analyzed the photocycles of the mutants with flash photolysis. The amino acid replacements caused profound changes to the photocycle of HeAR including acceleration of the decay of M intermediate, prolonging lifetime of intermediates in the later part of photocycle, and appearance of additional two intermediates which were evident in the photocycles of T164 mutants. These results suggest that although T164 and S165 are located at the far end of the photoactive center, these two amino acid residues are important for regulation of the HeAR photocycle. Moreover, the thermal stability of three-dimensional structure of HeAR and its mutants were investigated by small angle x-ray scattering (SAXS). In addition, the photocycles of HeAR reconstituted into lipid and HeAR in the presence of detergent were compared and the influence of the different environment on HeAR photocycle was investigated.","subitem_description_language":"en","subitem_description_type":"Abstract"},{"subitem_description":"細菌ロドプシン類タンパク質は7本のα-へリックスから構成され、発色団としてビタミンAアルデヒド（レチナール）を持つ光受容膜タンパク質であり、レチナールに吸収された光エネルギーの生体エネルギーへの変換や、外界の光情報の受容に関わっている。細菌ロドプシン類タンパク質は、生体膜中での立体構造が明らかにされた最初の膜タンパク質であり、モデル膜タンパク質として、詳細に研究され、それらの機能について、個々のアミノ酸レベルで、その分子機構が議論されている。本研究では、センサリーロドプシンII（SRII）と、中国内モンゴルの塩湖から我々のグループによって発見されたHalorubrum sp. ejinoor の持つアーキロドプシン(HeAR)について、新たなアミノ酸変異体を作成し、その光化学的性質を調べ、これまで分かっていなかったアミノ酸の働きを明らかにした。SRIIは、細菌が青緑光から逃げる負の走光性の光受容体である。本研究では、バクテリオロドプシン（BR）のプロトンポンプ機能に重要なアミノ酸残基であるアスパラギン酸（D）115に対応するSRIIのD103の変異体を作成した。SRIIのD103をアスパラギン（D103N）やグルタミン酸（D103E）に置換した変異体を作成し、その光反応を調べた。その結果、変異体では、M中間体の崩壊は速くなるが、光反応サイクル後半の中間体の寿命が長くなること、また、長寿命の副産物の生成が観察され、SRIIのD103は、SRIIの光反応サイクルの回転速度の調節に重要な役割を果たすことが分かった。HeARは、BRと同様に光駆動外向きプロトンポンプとして働く。ARのアミノ酸配列はBRとよく似ており、これまで、プロトンポンプ機能に必須であることが明らかになっているアミノ酸は全て保存されていた。しかし、ARだけに共通して保存されているアミノ酸があることに注目し、これらのアミノ酸（N末より164番目のトレオニン；T164と165番目のセリン；S165）をアラニン（A）に置換した変異体、T164A、S165AとT164A/S165Aを作製し、それらの光化学的性質を調べた。その結果、変異体では、M中間体の崩壊は速くなるが、光反応サイクル後半の中間体の寿命が長くなり、また、T164の変異では光反応中間体の数が増加した。発色団レチナールから離れた場所にあるにもかかわらず、HeARのT164とS165は、HeARの光反応サイクルの回転速度の調節に重要な役割を果たすことが分かった。また、HeARの立体構造の熱安定性をX線小角散乱法（SAXS）によって調べた。脂質に再構成したHeARと界面活性剤存在下でのHeARの光反応を比較検討し、タンパク質周辺環境の違いの影響についても調べた。","subitem_description_language":"ja","subitem_description_type":"Abstract"}]},"item_81_dissertation_number_13":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"甲第430号"}]},"item_81_identifier_registration":{"attribute_name":"ID登録","attribute_value_mlt":[{"subitem_identifier_reg_text":"10.15118/00009903","subitem_identifier_reg_type":"JaLC"}]},"item_81_subject_9":{"attribute_name":"日本十進分類法","attribute_value_mlt":[{"subitem_subject":"464","subitem_subject_scheme":"NDC"}]},"item_81_text_12":{"attribute_name":"学位の種別","attribute_value_mlt":[{"subitem_text_language":"ja","subitem_text_value":"課程博士"}]},"item_81_text_14":{"attribute_name":"報告番号","attribute_value_mlt":[{"subitem_text_language":"ja","subitem_text_value":"甲第430号"}]},"item_81_text_15":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_text_language":"ja","subitem_text_value":"博甲第430号"}]},"item_81_text_16":{"attribute_name":"研究科・専攻","attribute_value_mlt":[{"subitem_text_language":"ja","subitem_text_value":"工学専攻・先端生産システム工学コース"}]},"item_81_version_type_24":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorAffiliations":[{"affiliationNameIdentifiers":[],"affiliationNames":[{"affiliationName":""}]}],"creatorNames":[{"creatorName":"GENG, Xiong","creatorNameLang":"en"},{"creatorName":"耿, 兄","creatorNameLang":"ja"}],"familyNames":[{},{}],"givenNames":[{},{}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2019-06-25"}],"displaytype":"detail","filename":"A430.pdf","filesize":[{"value":"8.1 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"A430","objectType":"fulltext","url":"https://muroran-it.repo.nii.ac.jp/record/9955/files/A430.pdf"},"version_id":"47d2e83b-5483-449b-bf2e-d4edaa4dcb51"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2019-06-25"}],"displaytype":"detail","filename":"A430_summary.pdf","filesize":[{"value":"376.2 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"A430_summary","objectType":"abstract","url":"https://muroran-it.repo.nii.ac.jp/record/9955/files/A430_summary.pdf"},"version_id":"532800a0-ac8a-4457-89aa-c949272a73a7"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"doctoral thesis","resourceuri":"http://purl.org/coar/resource_type/c_db06"}]},"item_title":"Biophysical study of bacterial rhodopsins","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Biophysical study of bacterial rhodopsins","subitem_title_language":"en"},{"subitem_title":"細菌ロドプシン類タンパク質の生物物理学的研究","subitem_title_language":"ja"}]},"item_type_id":"81","owner":"18","path":["227"],"pubdate":{"attribute_name":"PubDate","attribute_value":"2019-06-25"},"publish_date":"2019-06-25","publish_status":"0","recid":"9955","relation_version_is_last":true,"title":["Biophysical study of bacterial rhodopsins"],"weko_creator_id":"18","weko_shared_id":-1},"updated":"2024-01-22T02:42:43.842825+00:00"}