2019 Fiscal Year Research-status Report
Encapsulation of alpha-synuclein oligomers into a protein cage to elucidate the dynamic behavior.
| Project/Area Number |
19K15695
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
Maity Basudev 東京工業大学, 生命理工学院, 特任助教 (60815421)
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| Project Period (FY) |
2019-04-01 – 2021-03-31
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| Keywords | Ferritin cage / Alpha-synuclein oligomer / High-speed AFM / Dynamics |
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| Outline of Annual Research Achievements |
Our proposed research is involved in isolating the amyloidogenic peptide, Alpha synuclein, into the confine environment of a ferritin cage and study the dynamic properties. So, we first established the observation of ferritin cage opening by high-speed AFM (submitted for publication). Then, we fused two different fragments of alpha-synuclein (residues 61-95 and 1-95) at the ferritin cage C-terminal. Native page, ESI and X-ray structure analysis suggest the successful encapsulation of 24 peptides into the cage. The high-speed AFM analysis suggests the formation of oligomer inside the cage. We now plan to study the details of oligomer structure and dynamics.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
The proposed research is proceeding more than we expected because our hypothesis was based on the preliminary experimental data. Based on the results we proceeded step by step. We already established the main basis of the work such as direct and real time visualization of the protein cage, disassembly mechanism and the formation of alpha-synuclein oligomer inside the cage. Therefore, the most difficult part is done now details analysis parts are left.
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| Strategy for Future Research Activity |
In our next research plan for the session 2020-2021, we will conduct the following studies and then present the results in the conferences and publish. (a) Summarizing the work and new design to encapsulate the full length (140 residues) of alpha-synuclein. (b) Determination of atomic structure by X-ray crystallography or cryo-EM. (c) Dynamic structure analysis of the encapsulated oligomer by high-speed AFM. (d) All-atom molecular dynamic simulations. (e) Cellular activity studies.
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| Causes of Carryover |
The budget 1,000,613 jpy is left for the next Fiscal year because we rescheduled the purchase of few equipments (Anion exchange, size exclusion Columns, HS-AFM parts etc). Therefore, we will use the budget in 2020 Fiscal year.
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