| Budget Amount *help |
¥15,860,000 (Direct Cost: ¥12,200,000、Indirect Cost: ¥3,660,000)
Fiscal Year 2018: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2017: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2016: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2015: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
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| Outline of Final Research Achievements |
Parkin, an E3 ubiquitin ligase, regulates mitophagy pathway by conjugation of ubiquitin chains on damaged mitochondria. Parkin is activated by phosphorylation and binding of phosphorylated ubiquitin. Although tertiary structures of parkin in the autoinhibited and activated states have been determined, its activation mechanism remains unclear, especially dynamic processes in activation are still elusive. In this study, we aim at elucidating structural dynamics of parkin by NMR to reveal the activation mechanism. For NMR measurements, a large amount of isotope-labeled protein is required, but parkin was obtained only in insoluble fractions in E. coli when we used M9 media. Here, we established a refolding method of parkin, in which insoluble parkin was solubilized by denaturant and the denaturant was removed gradually. The refolded parkin was monomeric and had ubiquitination activity.
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