Enhancement of thermostability of protein by reinforcement of subunit-subunit interaction
| Project/Area Number |
10650782
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Shinshu University |
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Principal Investigator |
HACHIMORI Akira Fac. of Textile Sci. and Tech. Shinshu University, Professor, 繊維学部, 教授 (30082811)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | 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 |
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| Research Abstract |
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
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Report
(3 results)
Research Products
(20 results)
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[Publications] 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)
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