2005 Fiscal Year Final Research Report Summary
INVESTIGATION OF THE MECHANISM OF AMYLOID FIBRIL FORMATION FROM PRESSURE EXPERIMENTS
| Project/Area Number |
16370054
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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 |
Structural biochemistry
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| Research Institution | KINKI UNIVERSITY |
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Principal Investigator |
AKASAKA Kazuyuki KINKI UNIVERSITY, SCHOOL OF BIOLOGY-ORIENTED SCIENCE AND TECHNOLOGY, PROFESSOR, 生物理工学部, 教授 (50025368)
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| Co-Investigator(Kenkyū-buntansha) |
FUJISAWA Masao KINKI UNIVERSITY, SCHOOL OF BIOLOGY-ORIENTED SCIENCE AND TECHNOLOGY, LECTURER, 生物理工学部, 講師 (20258065)
ANDO Yukio KUMAMOTO UNIVERSITY, SCHOOL OF MEDICINE, LECTURER, 医学部, 講師 (20253742)
TACHIBANA Hideki KOBE UNIVERSITY, FACULTY OF SCIENCE, ASSOCIATE PROFESSOR, 理学部, 助教授 (70126118)
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| Project Period (FY) |
2004 – 2005
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| Keywords | amyloid protofibril / hen lysozyme / pressure-jump NMR / high pressure fluorescence / pressure dissociation of protofibrils / activation volume for dissociation / transthyretin mutant / thermodynamic stability of transthyretin |
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| Research Abstract |
1. We have found, by using pressure-jump proton NMR spectroscopy, that the amyloid protofibrils of disulfide-deficient hen lysozyme are dissociated into monomeric species by applying pressure. The dissociation is fully reversible with pressure ; namely, upon lowering pressure, the freed monomeric proteins restart association into protofibrils.
2. The above phenomenon is studied in detail using Trp fluorescence under variable pressure and atomic force microscopy. The analysis of the experimental results have established that the association and dissociation of the disulfide-deficient hen lysozyme takes place by the linear polymerization mechanism, in which the monomeric protein is attached and detached to and from one end of the protofibril, respectively. The dissociation rate is strongly pressure-dependent. The analysis of the pressure dependence shows that the activation volume for the dissociation is negative (ΔV^<o【double plus】>=-50.5±1.60 ml mol^<-1>) and that the activation compressibility is also negative. These results have led us to conclude that the protofibril is a high-volume state, partial hydration of which leads to the activated state for dissociation.
3. Thermodynamic stability of the wild-type and V30M mutant of transthyretin is studied as a function of pressure using Trp fluorescence as monitor in the temperature range between 0 and 37℃. The stability extrapolated to 1 bar indicates that V30M mutant is less stable in the entire temperature range studied. The results confirms our earlier conclusion from high pressure NMR that the amyloid fiber formation of V30M mutant is correlated to its thermodynamic instability.
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