Enhancement of thermostability of protein by reinforcement of subunit-subunit interaction

通过加强亚基-亚基相互作用增强蛋白质的热稳定性

• 批准号: 10650782
• 负责人: HACHIMORI Akira
• 金额: $ 0.64万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10650782/
• 关键词: Inorganic pyrophosphatase; thermostability; site-directed mutagenesis; Thermophilic bacterium PS-3; Bacillus subtilus; protein structure; Biotechnology; subunit-subunit interaction; サブユニット間相互作用

In order to reveal the contribution of intra-subunit interaction on the thermostability of protein, inorganic pyrophosphatase (Ppase) from thermophilic bacterium PS-3 as a model protein was investigated and the following results were obtained.1. His-118 and His 125, which locate in helix A, are important for both trimer-trimer interaction and structural integrity, whereas Trp-143 is important structurally. The trimer-trimer interaction is absolutely necessary for the thermostability of the PS-3 PPase.2. Escherichia coli PPase is also thrmostable although its maximum temperature is 10 ℃ lower than that of PS-3 PPase. Thus we investigated the thermolabile PPase and we found it in Bacillus subtilis. We revealed the primary structure of B. subtilis PPase from the genomic DNA and found that not only the primary structure including the number of amino acid residues but also the quaternary structure are different from the soluble PPase so far studied. We concluded B. subtilis PPase is a membe … More r of new PPase family.3. We revealed the primary structure of Bacillus stearothermophilus PPase through the study of both Edman degradation and genomic DNA. We found that it is completely identical with that of PS-3 PPase except for the shortage of 2 amino acid residues at C-terminus. Site-directed mutagenesis revealed that Tyr-46 and Tyr-140 are very important for the maintenance of correct structure of active site pocket.4. PCR-mutagenesis classified the roles of 10 proline residues of PS-3 PPase into three groups; P39 and P69 have no contribution for the structural integrity. P14, P43 P69 and P116 are important for the secondary structure. P72, P100, P104 and P142 are important for the structural integrity.5. Ser-89 are important for the structural integrity of PS-3 PPase, and its replacement with Glu and Asp enhanced the thermostability of the enzyme by 10 ℃.6. The replacement of Arg-129, which locates in helix A necessary for trimer-trimer interaction, enhanced the thermostability of enzyme by 10 ℃. Less

为了揭示亚基间相互作用对蛋白质热稳定性的影响，以嗜热菌PS-3的无机焦磷酸酶（Ppase）为模型蛋白，研究了其热稳定性. His-118和His-125位于螺旋A中，对三聚体-三聚体相互作用和结构完整性都很重要，而Trp-143在结构上很重要。三聚体-三聚体相互作用对PS-3 PPase的热稳定性是绝对必要的.大肠杆菌PPase也是热稳定的，但其最高温度比PS-3 PPase低10 ℃。因此，我们研究了不耐热的PPase，我们发现它在枯草芽孢杆菌。我们揭示了B的一级结构。从基因组DNA中提取了枯草杆菌PPase，发现不仅包括氨基酸残基数目在内的一级结构，而且四级结构都与迄今为止研究的可溶性PPase不同。我们得出结论B。枯草杆菌PPase是一种 ...更多信息 新PPase家族的r.通过对Edman降解和基因组DNA的研究，揭示了嗜热脂肪芽孢杆菌PPase的一级结构。结果表明，该酶与PS-3 PPase的C-端氨基酸残基完全相同，但缺少2个氨基酸残基。定点突变表明Tyr-46和Tyr-140对维持活性位点袋的正确结构非常重要. PCR-诱变将PS-3 PPase的10个脯氨酸残基的作用分为三组，P39和P69对结构完整性没有贡献。P14、P43、P69和P116是重要的二级结构。P72、P100、P104和P142对结构完整性有重要作用. Ser-89对PS-3 PPase的结构完整性有重要作用，Glu和Asp的取代使酶的热稳定性提高了10 ℃.精氨酸129的取代使酶的热稳定性提高了10 ℃。少

项目成果

野村隆臣: "蚕絹糸腺リボソームの調整とその性質"日蚕雑. 40. 311-318 (1998)

Takaomi Nomura：“蚕丝腺中核糖体的调节及其特性”Nissho Zas。 40. 311-318 (1998)

Shintani, T.,: "Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis. Evidence for a new family of enzyme."FEBS Lett.. 439. 263-266 (1998)

Shintani, T.，：“枯草芽孢杆菌中独特的无机焦磷酸酶的克隆和表达。新酶家族的证据。”FEBS Lett.. 439. 263-266 (1998)

Sato, T., Shinoda, H., H., Hachimori, A., Irie, M., Samejima, T.: "Primary structure, expression and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothermophilus"J. Biochem.. 125. 681-686 (1999)

Sato, T.、Shinoda, H.、H.、Hachimori, A.、Irie, M.、Samejima, T.：“嗜热脂肪芽孢杆菌无机焦磷酸酶的一级结构、表达和定点突变”J。

Uchiumi, T., Hori, K., Nomura, T. and Hachimori, A.: "Replacement of L7/L12.L10 protein complex in Escherichia coli ribosomes with the eukaryotic counterpart changes the specificity of elogation factor binding."J. Biol. Chem.. 274. 27578-27582 (1998)

Uchiumi, T.、Hori, K.、Nomura, T. 和 Hachimori, A.：“用真核对应物替换大肠杆菌核糖体中的 L7/L12.L10 蛋白复合物改变了延伸因子结合的特异性。”

T.Satoh: "Primary structure,expression,and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothevmophilus" J.Bischem.125. 48-57 (1998)

T.Satoh：“嗜脂肪芽孢杆菌无机焦磷酸酶的初级结构、表达和定点诱变”J.Bischem.125。