| Project/Area Number |
13680752
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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 | Osaka University of Pharmaceutical Sciences |
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Principal Investigator |
ISHIDA Toshimasa Osaka University of Pharmaceutical Sciences, Pharmacy, Professor, 薬学部, 教授 (00111021)
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| Co-Investigator(Kenkyū-buntansha) |
MINOURA Katsuhiko Osaka University of Pharmaceutical Sciences, Pharmacy, Assistant, 薬学部, 助手 (10278591)
TOMOO Koji Osaka University of Pharmaceutical Sciences, Pharmacy, Lecturer, 薬学部, 講師 (70257898)
IN Yasuko Osaka University of Pharmaceutical Sciences, Pharmacy, Assistant, 薬学部, 助手 (50257896)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2003: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | tau protein / formation mechanism of tangle / microtubule-binding domain / 3D-structure / X-ray analysis / NMR / Spectroscopic analysis / molecular modeling / タングル形式 |
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| Research Abstract |
1)X-ray crystal structure analysis of microtubule-binding domain
To elucidate 3D structure of tau protein, we attempted various crystallizations of tau and its fragments. Consequently, we succeeded in the crystallization of tau microtubule-binding domain (MBD) as GST fusion protein, which were prepared as recombinant by gene expression from E.coli. The crystal data are : tetragonal, space group P4_32_12, a=b=92.24Å, c=57.68Å. X-ray diffraction data up to 2Å resolution were collected by synchrotron of Spring 8. The crystal structure was solved by the molecular replacement method using the 3D structure of GST. After several structure refinement, the present R factor is 0.258. Although the MBDs formed dimeric structure, they showed large conformational flexibility and their atomic positions were not determined. This is in contrast with the rigid 3D structure of GST. Since the MBD constructs the core structure of tau PHF, the present result indicates the close relationship between the confo
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rmational flexibility of MBD and PHE formation. We are now in progress in analyzing the crystal structure more accurately.
2)Analyses of solution structure of MBD
MBD is composed of three-or four-repeated structure, where each repeat peptide (named R1 -R4) consists of 31 or 32 amino acid residues. To elucidate the conformational flexibility of each repeat peptide, the solution structures of R1 -R4 in TEE solution were analyzed by the combination of NMR and model building. Consequently, the the structures of R1, R2 and R4 peptides showed α-helical structure of amphipathic distribution of respective amino acid residues. In contrast, R3 took both of extended and α-helical structures of amphipathic behavior. This result suggest the molecular association of MBD through alternative hydrophilic and hydrophobic interactions.
3)Investigation of MBD interactions and model of repeat-structure-dependent PHF formation
The association of neighboring MBDs were investigated by ThS fluorescence, light scattering and surface plasmon resonance analyses, and electromicroscopy. On the basis of these results, we proposed the first model of repeat structure-controlled step-by-step filament formation of 4RMBD. This model would be helpful upon considering rational approaches to prevent tau deposition in AD Less
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