| 研究実績の概要 |
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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| 今後の研究の推進方策 |
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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