Characterization of the aggregation propertis of amyloid core in the TDP-43 C-terminal fragments

• Main Authors: Chun-Hao Liu, 劉君浩
• Other Authors: 陳長謙
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/57248370187337331522

碩士 === 國立臺灣大學 === 化學研究所 === 100 === It has been known that the core sequence of cleaved peptides or proteins provides molecular recognition as well as self-assembly driving force during amyloid fibrils formation. In 2010, the peptide fragment D1 (287-322) from the C-terminal domain of TDP-43 has been demonstrated to form amyloid fibrils in phosphate buffer. To further characterize the amyloid core in this fragment, we sequentially synthesize the truncated peptides and confirm the amyloid fibrils formation by Circular Dichroism (CD), Transmission Electronic Microscopy (TEM), Raman spectroscopy and Thioflavin T (ThT) assay. We had found that only the peptides containing the sequence (307-322) may undergo a conformational change from the random coil to a β-sheet rich structure when compared to the shorter peptides (312-322, 307-317). Moreover, the hydrophobicity, average flexibility and beta-sheet predictions have been applied to confirm the biophysical properties of these peptides. According to CD results, amyloid peptides having increasing intensity in β-sheet have been carefully compared. The core sequence shows higher increasing rate compared to the longer amyloid peptides. In order to gain insight into the properties of the amyloid core sequence, three glycines in the peptide TDP307-322 has been replaced to proline, TDP307-322 G3308P3, as proline had been reported to block the β-sheet structure. We expect to see the proline can block the β-sheet structure formation and indirectly inhibit amyloid fibrils formation. To our surprise, the triple-proline we introduced somehow make the peptide to generate a strong poly(L-proline) Ⅱ (PPⅡ)-helical conformation in CD spectra, and as a matter of fact, the wild-type TDP307-322 was observed to contain PPⅡconformation during the conformational change as well. Therefore, we postulate the residues in 307-312 may have significant impact of aggregation and fibril formation. Here, we replace one and two proline substitutions in between zero and three in the core sequence to see the perturbation of amyloid fibrils formation in different extent. What we have found is that the different amounts of proline substitutions demonstrate delicate behavior in tuning the amyloid fibrils formation. The PPⅡ helix conformation might play an important role in kinetically controlling the intermediate state toward β-sheet structure during amyloid fibril formation. If we can find a way to redirect the aggregation pathway based on the possible intermediate state and control the amyloid properties during aggregation, it is possible to reverse the situation which related to oligomer toxicity, membrane pore formation, or some other defect in amyloidogenesis.