2015 Fiscal Year Research-status Report
Study on cold and heat denaturation of amyloid fibrils of intrinsically disordered alpha-synuclein
| Project/Area Number |
15K18518
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| Research Institution | Osaka University |
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Principal Investigator |
李 映昊 大阪大学, たんぱく質研究所, 講師 (70589431)
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| Project Period (FY) |
2015-04-01 – 2017-03-31
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| Keywords | Alpha-synuclein / Amyloid fibril / Cold denaturation / Heat denaturation / Kinetics / Protein aggregation / Protein misfolding / Thermodynamics |
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| Outline of Annual Research Achievements |
The stability of amyloid fibrils remains poorly understood. Thus, I studied the conformational stability of amyloid fibrils of alpha-synuclein (aSN) against the temperature change as a model system. I examined cold and heat denaturation of aSN amyloid fibrils prepared at various temperature (298, 310, 323, 333, and 343 K). At all temperature, the formation of typical amyloid fibrils was confirmed by ThT assay, far-UV CD spectra, and AFM images. As temperature increased from 298 to 383 K, all types of fibrils heat-denatured to monomers from approximately 343 K. Decreasing temperature from 298 to 273 K caused cold denaturation of fibrils to monomers. Intriguingly, the stability of aSN fibrils depended on temperature of preparation of aSN fibrils. aSN fibrils generated at higher temperature showed higher stability at high and low temperature than fibrils formed at lower temperature. The stability of aSN fibrils prepared at higher temperature against chemical stress also was higher than that of fibrils formed at lower temperature. Kinetics of cold and heat denaturation of individual aSN fibrils with and without a denaturant were also traced. As temperature where aSN fibrils grow became higher, fibrils showed faster denaturation. Even with a chemical denaturant, similar trends of denaturation kinetics were observed. Overall, the stability of aSN amyloid fibrils depended on growing temperature of fibrils.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The study on cold and heat denaturation of aSN amyloid fibrils has well been performed as I planned without problems. I clearly revealed that aSN amyloid fibrils denatured to soluble monomers at high and low temperature and growing temperature influenced largely the conformational stability of aSN amyloid fibrils. Several approaches will be performed for elucidating factors which control the conformational stability of aSN fibrils against temperature change in the following section.
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| Strategy for Future Research Activity |
In order to elucidate the mechanism and factor for cold and heat denaturation of aSN amyloid fibrils, following experiments will be performed.
1. Checking reversibility to ensure the thermodynamic analysis: after cold and heat denaturation of fibrils, temperature will be set to the original temperature, and the reformation of fibrils will be monitored.
2. Conformational analysis of aSN amyloid fibrils: (1) The secondary structure of aSN amyloid fibrils prepared at distinct temperature will be examined by Fourier transform infrared spectrometry. (2) The morphology of each aSN amyloid fibril will be analyzed using the high-resolution EM at the single fibril level. (3) The core regions of individual aSN fibrils will be examined by proteolysis and mass spectrometry.
In order to enhance pathological and biological relevance of the present study, cold and heat denaturation behaviors of amyloid fibrils of aSN mutants as well as roles of the membrane and crowding effect on the conformational stability of aSN fibrils will be also investigated.
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| Causes of Carryover |
実験の進行状態、結果そして効率の良い研究費の使用を考慮し、3月末に試料を購入する必要が生じたた。
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| Expenditure Plan for Carryover Budget |
残額「101,368円」に関しては、future planで計画しているmembraneを摸倣したvesicleを作製するための材料および試薬の購入に主に使用する予定であり、既に注文してある。
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[Journal Article] Physicochemical Nature of Interfaces Controlling Ferredoxin NADP+ Reductase Activity through Its Interprotein Interactions with Ferredoxin2015
Author(s)
Kinoshita Misaki, Kim Ju Yaen, Kume Satoshi, Sakakibara Yukiko, Sugiki Toshihiko, Kojima Chojiro, Kurisu Genji, Ikegami Takahisa, Hase Toshiharu, Kimata-Ariga Yoko, Lee Young-Ho
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Journal Title
Biochimica et Biophysica Acta-Bioenergetics
Volume: 1847
Pages: 1200-1211
DOI
Int'l Joint Research / Acknowledgement Compliant
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