| Project/Area Number |
09470492
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Yokohama National University |
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Principal Investigator |
UESUGI Seiichi Yokohama National University, Faculty of Engineering, Professor, 工学部, 教授 (70028851)
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| Co-Investigator(Kenkyū-buntansha) |
KURIHARA Yasuyuki Yokohama National University, Faculty of Engineering, Research Associate, 工学部, 助手 (80202050)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥11,700,000 (Direct Cost: ¥11,700,000)
Fiscal Year 1999: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1998: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1997: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Keywords | proto-oncogene / DNA binding protein / transcription factor / c-Myc / Max / mutant protein / DNA-porotein interaction / structure / 蛋白質 |
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| Research Abstract |
Max Protein regulates the function of a proto-oncogene product, Myc, by formation of a heterodimer with Myc or a homodimer with itself and binding to DNA. We established a procedure for preparation of Max and two types of its DNA binding domains by genetic engineering techniques and examined their structure and properties. Max proteins were in equilibrium between a monomer and a dimer in solution. The dimer content increasesd with increasing protein concentration and decreasing temperature. Increase in α-helical content of the protein simultaneously accompanied the dimer increase. Upon addition of a duplex DNA containing the specific recognition base sequence, the α-helical content increased further with increasing DNA concentration. When a DNA duplex containing a sequence different in the central two base pairs was used, the α-helical content increase was smaller. A strictly specific recognition of the cognate sequence was observed at high temperature. The biological activity of Max is modulated by phosphorylation of Ser residues in the N-terminal region. We prepared mutant proteins where one or two of the Ser residues were replaced with Asp residue(s) as a mimic of the phosophorylated Max and examined DNA binding ability of the mutant proteins. The mutant protein-DNA complexes showed lower stability and the complex of the mutant protein with double mutation showed the lowest stability. We also developed a synthetic method for obtaining phosphorylated Max by chemically joining a phosphorylated peptied synthesized chemically and the remaining part of protein prepared by genetic engineering.
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