| Project/Area Number |
11680665
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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 |
Biophysics
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| Research Institution | Kitasato University |
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Principal Investigator |
KODERA Yoshio KITASATO UNIVERSITY, SCHOOL OF SCIENCE, LECTURER, 理学部・生体分子動力学研究室, 講師 (60265733)
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| Co-Investigator(Kenkyū-buntansha) |
KOHNO Toshiyuki MITSUBISHI KASEI INSTITUTE OF LIFE SCIENCES, CHEEF RESEARCHER, 構造生物学研究ユニット, 主任研究員
KOMATSU Hiroyoshi KITASATO UNIVERSITY, SCHOOL OF ALLIED HEALTH SCIENCE, ASSISTANT, 医療衛生学部, 助手 (30170377)
MAEDA Tadakazu KITASATO UNIVERSITY, SCHOOL OF SCIENCE, PROFESSOR, 理学部, 教授 (90265728)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | HIV-2 / nucleocapsid protein / three-dimensional structure / NMR / NCp8 / RNA-bainding property / ヌクレオキャップシドタンパク質 / RNA認識 / ヌクレオキャプシドタンパク質 |
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| Research Abstract |
1. Structure-activity relationships of NCp8-f1/N11A
The NC protein, NCp8, of HIV-2 is a 49 AA peptide containing two zinc fingers connected by seven amino acid residues called "basic amino acid cluster". It has been shown that the N-terminal zinc finger flanked by basic amino acid cluster is the minimal active domain. In 1998, three-dimensional structure of a 29 amino acid peptide (NCp8-f1) including the minimal active domain was determined. The basic amino acid cluster was well-defined with a loop-like conformation. The conformation of the cluster is likely to be stabilized by the hydrogen bonds between the cluster and the zinc finger: between Arg^<27> 0 and Asn^<11> N_sH (Y. Kodera et al., Biochemistry, 37, 1998).
In the present study, the RNA-binding property of NCp8-f1 derivative with Asn^<11> replaced by Ala (NCp8-f1/N11A) was analyzed using ultraviolet cross-linking assay and the three-dimensional structure of this peptide was determined by ^1H NMR spectroscopy with simulated annealing calculations. The RNA-binding property of NCp8-f1/N11A was different from that of NCp8-f1. The conformation of the basic amino acid cluster was not defined. These results suggest mat the conformation of the basic amino acid cluster is important for specific RNA binding.
2. Determination of the three-dimensional structure of NCp8-f2
NCp8-f2 is a 27 amino acid peptide, which includes the C-terminal zinc finger and the basic amino acid cluster. The three-dimensional structure of NCp8-f2 was determined. The structure-activity relationships are discussed on the basis of the comparison of this structure with those of NCp8-f1, NCp7-f2 and NCp10.
3. Preparation of uniformly ^<15>N-enriched NCp8
NCp8 uniformly enriched with ^<15>N, which is necessary to determine the three-dimensional structure of the NCp8 and the NCp8-RNA complex, was expressed in E. coli cells and purified using a ubiquitin fusion protein system. 5mg of this sample has been prepared.
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