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

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

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