| Project/Area Number |
08559009
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
広領域
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| Research Institution | Nagoya University |
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Principal Investigator |
GO Mitiko Nagoya University, Division of Biological Science, Professor, 大学院理学研究科, 教授 (70037290)
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| Co-Investigator(Kenkyū-buntansha) |
HOJO Hironobu Osaka City University, Bioapplied Chemistry, Lecturer, 工学部, 講師 (00209214)
YURA Kei Nagoya University, Division of Biological Science, Research Associate, 大学院理学研究科, 助手 (50252226)
NOGUTI Tosiyuki Nagoya University, Division of Biological Science, Associate Professor, 大学院理学研究科, 助教授 (90172775)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥10,300,000 (Direct Cost: ¥10,300,000)
Fiscal Year 1997: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1996: ¥6,600,000 (Direct Cost: ¥6,600,000)
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| Keywords | making protein soluble / barnase / mini-protein / reverse transcriptase / amino acid replacement / domain fusion / protein evolution / module |
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| Research Abstract |
In order to know structure and function of gigantic protein and protein complex, a method to cut and take out stable partial structure of protein and a method to make the partial structure soluble are needed. Our purpose is to develop a design method to take out partial structure of protein in a soluble and stable from. We intended further to verify that the designed partial structure forms soluble and stable structure in solvent. In process of molecular evolution, a fusion of domain or module occurred frequently. We showed that in reverse transcriptase, on the occasion of RNaseH domain fusion, at least 4 amino acid replacements occurred in adopting to the atomic interactions at domain contact. We applied the same method to make a partial protein structure soluble. Previously we found a barnase-like domain in RNA polymerase. We planed to cut out the barnase-like domain from RNA polymerase and designed a soluble form of isolated barnase-like domain. Then we did a chemical synthesis of the sequence. For getting soluble barnase-like domain, we established a method to replace amino acid residues using an evolutionary replacement pattern of amino acid in domain fusion. This method is applicable for the proteins whose three-dimensional structures are unknown. Also, we designed a mini-barnase by removing a module from barnasr, performed molecular dynamics and evaluated its solubility and stability by free energy calculation. Mini-barnase lacking 26 amino acid residues was chemically synthesized. We measured CD and NMR of designed mini-barnase. It was dissolved into water and took a stable structure similar to the conformation of a barnase except the excised region. Through this research, we developed a method that is useful in cutting and taking out a part of protein in soluble and stable form.
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