Studies on the Signal Transduction Mechanism by Gas Sensor Proteins

气敏蛋白信号传导机制的研究

• 批准号: 13470480
• 负责人: UNO Tadayuki
• 金额: $ 3.46万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470480/
• 关键词: Gas Molecules; Sensor Protein; Resonance Ramen Spectroscopy; Transcription Factor; Heme Protein; ガスセンサー; シグナル伝達

In this research, various mutants of two gas sensor proteins, DOS and CooA, were prepared in order to reveal signal transduction mechanism triggered by gas molecules such as oxygen and CO. Firstly, axial ligands of Dos were identified since this protein was found to contain six-coordinate low-spin heme. On the basis of structural information of FixL which is homologous to Dos, Met 95 of Dos was replaced by Gln, and absorption and resonance Raman spectra were measured. This mutant was found to contain five-coordinate high-spin heme, and the Met95 was established to be one of the axial ligand of Dos. On the other hand, mutation was introduced to amino acid residues in close proximity of the heme cofactor in order to reveal the DNA binding mechanism of CooA, which is a transcription factor and senses CO partial pressure. His77 residue was replaced by Asn, Ser, and Arg which have different polarity, and identity of the axial ligand replaced by CO was investigated. These mutations were found to result in the formation of five-coordinate heme, but bound v(Fe-CO) stretching Raman band was insensitive to the mutations. Thus, CO was suggested to replace Pro2 residue trans to the His77 ligand. In addition, resonance Raman spectra were measured for the mutants Cys105, Leu112, Ile113, Ala114, Leu116, Gly117, Arg118, Leu120, Tnr121, Arg125, and Met131, which reside on the center helix of CooA and are supposed to concern in the signal transduction. The v(Fe-CO) stretching fiequency were found to shift greatly for L116Q, G117N, and L120Q, and these residues were suggested to locate closely to the bound CO gas. Furthermore, footprint analysis revealed that DNA binding activity was lost in these mutants.

本研究制备了两种气体传感器蛋白DOS和CooA的各种突变体，以揭示氧气和CO等气体分子触发的信号转导机制。首先，由于DOS蛋白含有六坐标低自旋血红素，因此鉴定了DOS的轴向配体。根据与Dos同源的FixL的结构信息，用Gln代替Dos的Met 95，测量吸收和共振拉曼光谱。发现该突变体含有五坐标高自旋血红素，并确定Met95是Dos的轴向配体之一。另一方面，在血红素辅助因子附近的氨基酸残基上引入突变，以揭示CooA的DNA结合机制，CooA是一种感知CO分压的转录因子。His77残基被不同极性的Asn、Ser和Arg取代，并被CO取代的轴向配体的身份进行了研究。这些突变导致五坐标血红素的形成，但结合的v（Fe-CO）拉伸拉曼带对突变不敏感。因此，CO被认为取代了Pro2残基转到His77配体上。此外，对位于CooA中心螺旋上可能与信号转导有关的突变体Cys105、Leu112、Ile113、Ala114、Leu116、Gly117、Arg118、Leu120、Tnr121、Arg125和Met131进行了共振拉曼光谱测定。在L116Q、G117N和L120Q中，v（Fe-CO）的拉伸频率发生了较大的位移，这些残基可能与CO结合气体紧密相关。此外，足迹分析显示，这些突变体的DNA结合活性丧失。

项目成果

Tadayuki Uno: "Cysteine Thiolate Coordination in the Ferrous CO Complex of an Engineered Cytochrome b562"J.Am.Chem.Soc.. 123. 2458-2459 (2001)

Tadayuki Uno：“工程化细胞色素 b562 的亚铁 CO 复合物中的半胱氨酸硫醇盐配位”J.Am.Chem.Soc.. 123. 2458-2459 (2001)

Tadayuki Uno, Akihiro Yukinari, Yoko Moriyama, Yoshinobu Ishikawa, Yoshrikazu Tomisugi, James A. Bianmgan, and Anthony J. Wilkinson: "Engineering a ligand Binding Podcet into a Four Helix Bundle Protein Cytochrome b562"J. Am. Chem. Soc.. 123. 512-513 (200

Tadayuki Uno、Akihiro Yukinari、Yoko Moriyama、Yoshinobu Ishikawa、Yoshikazu Tomisugi、James A. Bianmgan 和 Anthony J. Wilkinson：“将配体结合 Podcet 设计成四螺旋束蛋白细胞色素 b562”J。

Tadayuki Uno: "Engineering a Ligand Binding Pocket into a Four Helix Bundle Protein Cytochrome b562"J.Am.Chem.Soc.. 123. 512-513

Tadayuki Uno：“将配体结合袋设计成四螺旋束蛋白细胞色素 b562”J.Am.Chem.Soc.. 123. 512-513

Tadayuki Uno: "Cysteine Thiolate Coordination in the Ferrous CO Complex of an Engineered Cytochrome b562"J.Am.Chem.Soc.. 123. 2458-2459

Tadayuki Uno：“工程化细胞色素 b562 的亚铁 CO 复合物中的半胱氨酸硫醇盐配位”J.Am.Chem.Soc.. 123. 2458-2459

Tadayuki Uno: "Engineering a Ligand Binding Heme Pocket into a Four Helix Bundle Protein Cytochrome b562"J.Am.Chem.Soc.. 123. 512-513 (2001)

Tadayuki Uno：“将配体结合血红素口袋设计成四螺旋束蛋白细胞色素 b562”J.Am.Chem.Soc.. 123. 512-513 (2001)