| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Protein aggregation has become one of the serious problems in the fields of biotechnology, medicine, and life science. In this study, we have studied basic mechanism of insoluble protein aggregation in vivo and in vitro by using various protein molecular-scientific techniques.
1. Characterization of folding intermediate of triose phosphate isomerase(TIM)
It has been elucidated that the folding intermediate of dimeric enzyme TIM was a partially structured monomeric species by NMR spectroscopic analysis.
2. Solubilization of inclusion body of thermostable Mn-catalase
Insoluble aggregations (inclusion body) were formed when Mn-catalase was overexpressed in E. coli. However, it has been elucidated that co-expression of molecular chaperones (GroEL, GroES, DnaK, DnaJ, and GrpE) overcome the situation, and the correct folded active enzyme was obtained efficiently.
3. Structural changes and amyloid-like fibril formation of GroES and α synuclein
Incubated chaperonin GroES, a heptameric protein, at a high protein concentration for several days in the presence of Gdn-HCl, global conformational changes occurred and formed insoluble amyloid-like fibrils, which has been elucidated by Thioflavin-T staining and the electron microscopy. In addition, it was observed that compaction of natively unfolded α synuclein by increasing ionic strength led to amyloid fibril formation readily. These results gave a significant insight on mechanism of conformational changes of various proteins related to insoluble aggregations and amyloid-like fibril formation.
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