| Project/Area Number |
06558100
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biophysics
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| Research Institution | Nagoya University |
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Principal Investigator |
GO Mitiko Nagoya University, Biology, Professor, 大学院・理学研究科, 教授 (70037290)
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| Co-Investigator(Kenkyū-buntansha) |
KUWAJIMA Kunihiro University of Tokyo, Physics, Associate Professor, 大学院・理学系研究科, 助教授 (70091444)
HOJO Hironobu Osaka city University, Bioapplied Chemistry, Lecturer, 工学部, 講師 (00209214)
NOGUTI Tosiyuki Nagoya University, Biology, Associate Professor, 大学院・理学研究科, 助教授 (90172775)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥13,000,000 (Direct Cost: ¥13,000,000)
Fiscal Year 1995: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1994: ¥8,100,000 (Direct Cost: ¥8,100,000)
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| Keywords | mini-barnase / module / RNase activity / molten globule / protein design / solid-phase method / segment condensation / thioester / タンパク質フォールディング |
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| Research Abstract |
Our purpose is to establish method of protein design based on module units. Our aims are twofold : (1) design of stable mini-proteins by deletion of module from a native protein and analysis of its three-dimensional structure, and (2) design of mini-proteins having function of its mother protein, chemical synthesis and study of its function and three-dimensional structure. The followings are the main results obtained during the two-years research project.
1) Design of a basic structure of mini-protein and evaluation of its conformational stability : We found that a mini-barnase designed by deletion of module M1 or M2 from barnase is a suitable candidate for designed mini-protein. We determined the amino acid replacements which were necessary to stabilize the mini-barnase by molecular dynamics calculation and evaluation of free energy change.
2) The chemical synthesis of the designed mini-barnase : We synthesized the mini-protein by using the procedure developed by Hojo et al. Partially p
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rotected peptide thioesters corresponding to two peptides and partially protected peptide were prepared via a solid-phase method. For the introduction of residues at ten sites, ^<15>N-labeled amino acid derivatives were used. The thee peptide segments were successively condensed by the activation of thioester groups by silver ions to give a protected form of mini-barnase.
3)Structure and function of the mini-barnases : On the basis of the chemical shift of the ^<15>N-labeled amino acids the mini-barnase without M2 was found to have a stable conformation similar with native barnase. The mini-barnase without M1 made a complex with module M1 and the complex had RNase activity.
In conclusion, we were successful in design of mini-proteins by deletion of one module in the sense that the designed proteins were soluble and stable. Also, a method of chemical synthesis of mini-protein was developed. The determination of the three-dimensional structure of the mini-barnases by NMR measurements and development of methods for design of mini-barnase with enzymatic activity are left for further study. Less
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