Comparison of Molecular Structures of Protofibrils and Mature Fibrils Formed by Residues 113-127 of Syrian Hamster Prion Protein
Date Issued
2009
Date
2009
Author(s)
Cheng, Hsin-Mei
Abstract
Amyloid fibrils are ordered aggregates of misfolded proteins, which associated with many neurodisorder diseases. Transmissible spongiform encephalopathy (TSE) is one of these fatal diseases. It is suggested that TSE is caused by the conversion of prion protein from its normal cellular form (cellular prion protein, PrPC) to the disease-specific form (scrapie prion protein, PrPSc).The presence of PrPSc will induce the misfolding of other PrPC to PrPSc, existing in the form of amyloid fibrils and damaging the nervous system of the victims. To elucidate the mechanism and pathways of fibril formation and to find the therapy, it is very important to analyze the molecular structure of amyloid fibrils. However, because amyloid fibrils are insoluble in most buffers and are non-crystalline, it is difficult to use conventional experimental techniques such as solution-state NMR or X-ray to obtain the detailed structure of amyloid fibrils. Solid state NMR spectroscopy is a unique method that can provide high-resolution, site-specific structural constraints for amyloid fibrils. In this thesis, we report the results of ThT fluorescence, TEM, AFM, FTIR, and solid-state NMR (SSNMR) data for protofibrils and mature fibrils formed by residues 113-127 of the Syrian hamster prion protein (SHaPrP113-127, Ac-AGAAAAGAVVGGLGG-NH2). We found that after purification, SHaPrP113-127 peptides have already had short fibrillar structure, and also an enhanced fluorescence upon binding to ThT. These properties confirm that the SHaPrP113-127 peptides right after purification are in protofibrillar state. After incubation, the length of the mature fibrils becomes longer and the intensity of the ThT fluorescence becomes significantly enhanced. The mature fibrils in general have a length over 300 nm, a width of 12 nm, and a height of 1 nm. From the chemical shift and the linewidth data obtained from SSNMR measurements, the β-sheet structure formed in both protofibrils and mature fibrils have significant ordered structure from the residue 115 to 120. Our data reveal that the molecular structure of mature fibrils adopts the motif of steric zipper, whereas, the structure of protofibrils dose not. From the FTIR spectra, the β-strands within each layer are anti-parallel. Consequently, a molecular model for the fibrils was constructed by molecular dynamics simulations incorporated with structural constraints obtained from solid-state NMR measurements. We suppose that the process of fibril formation takes the following pathway. First, monomeric SHaPrP113-127 molecules self-assemble into single layer that is predominated by anti-parallel β-sheet. After that, two layers mate tightly and form the steric zipper by the hydrophobic interaction of side chains.
Subjects
prion protein
amyloid fibril
solid-state NMR
protofibril
Type
thesis
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