パーツ並べ変え蛋白質のX線解析による新しいバイオインフォマティクス基盤の確立

利用部分重排蛋白质的 X 射线分析建立新的生物信息学平台

• 批准号: 15657036
• 负责人: 片柳 克夫
• 金额: $ 1.92万
• 依托单位: Hiroshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Exploratory Research
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15657036/
• 关键词: X線構造解析; パーツ並べ替えタンパク質; モルテングロビュール状態; アンフィンセンのドグマ; 進化分子工学; タンパク質構造構築原理; 構造ゲノム科学; 超高分解能結晶

本研究では,タンパク質の二次構造単位領域(パーツ)でポリペプチド鎖を入れ替えたタンパク質の結晶構造を世界に先駆けて解明した。柳川が以前バルナーゼについて,二次構造(Sシリーズ)とモジュール(Mシリーズ)の観点からそれぞれ6つのパーツにわけ,すべての組み合わせに対するパーツ並べ替えタンパク質46種類を作成した中から,比較的立体構造や活性を持っているS2543とS2354H102A(S2354は構造が取れなくH102A変異体にして構造をとったもの)について,大腸菌系における大量培養・精製,結晶化,構造解析を行った。S2543についてはANSを用いた蛍光実験からモルテングロビュール状態と考えられていたが,我々は1.5Åを超える高分解能の結晶を得,さらに放射光実験で1.1Å超高分解能でのデータ収集に成功した。しかし重原子同型置換法による構造解析の結果,全長110アミノ酸残基に対し約10残基の電子密度の観測不可能な部分があった。これが揺らぎによるものか否かを決定付けるために,2年目は基質アナログである2'3'GMPとの複合体の結晶化・構造解析にも成功した。その結果,S2354は超遠心の実験から2量体と示唆されていたが,結晶構造でもC端側がDomain swapした2量体であった。ただし基質GMP有無で会合構造が大きく異なることがわかった。GMPS2354の場合はS2543よりさらに不安定で濃縮が困難であったが,改良を重ねた結果高濃度濃縮に成功し,微結晶析出までこぎつけた。いずれにせよ,これだけ大胆に1次配列を変えたにもかかわらず超高分解能での詳細構造が得られたことは,現在の構造ゲノム科学を支えるアンフィンセンのドグマに反する立体構造を詳細に解き明かしたことであり,進化分子工学やタンパク質構造構築原理などのバイオインフォマティクスに多大な貢献が期待できる結果である(論文作成中)。

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