Solid State NMR Study of Amyloid Fibrils Formed by Mutant Syrian Hamster PrP109-122 and Human PrP127-147
Date Issued
2010
Date
2010
Author(s)
Tang, Yun-Ju
Abstract
Abstract
Prion particles (prion) are proteinaceous infectious particles and it has been proved that there are two different structures exist for prion proteins. The normal cellular prion protein, denoted as PrPC, may convert into an abnormal scrapie cellular prion, denoted as PrPSc, through a process whereby a portion of its α-helical structure is refolded into β-sheet. The presence of PrPSc will catalyze the transformation of PrPC to PrPSc by the misfolding process. When PrPSc forms aggregate through hydrogen bonds and Van der Waals interactions, the amyloid fibril will be formed and cause the neurodegenerative disease. The transmissible spongiform encephalopathies (TSE) is one of the neurodegenerative diseases that caused by the amyloid fibril depositions. Different protein sequences may have different pathogenicities and distinctive amyloid fibril structures. Therefore, understanding structural conversion of amyloid fibril is very important. Owing to the low solublity and non-crystalline characteristics of amyloid fibrils, it is difficult to use conventional experimental techniques such as solution-state NMR and XRD to analyze the structure of amyloid fibrils. Hence, solid-state NMR is still the most suitable method to characterize structures of this kind.
In this study, we mutated the 117 position of Syrian hamster prion protein 109-122 fragment (SHaPrP109-122, A117I, Ac-MKHMAGAAIAGAVV-NH2) from Ala to Ile. The idea is to compare the structural difference induced by mutation on the aligned position of β-sheet in the native SHaPrP109-122. We have incubated the amyloid fibril formed by SHaPrP109-122, A117I successfully. We report the ThT fluorescence, TEM, AFM experiments to observe the formation of amyloid fibrils. From the TEM and AFM images, we have measured the matured fibrils about 24 nm in width, 1.1 nm in height and 380 nm in length. From the isotope-edited FTIR study, the fibril has an anti-parallel β-strand secondary structure and showed an obvious alignment at I117. The chemical shift and linewidth data obtained from ssNMR showed that β-sheet structure exists in the core region. The distance about 4.9±0.2Å between the two β layers is determined by fpRFDR-CT experiment. From the experimental data it has been inferred that the fibrils formed by A117I SHaPrP109-122 has the steric zipper structure. Finally, a molecular model for the fibrils was constructed by molecular dynamics simulations incorporated with structural constraints obtained from ssNMR measurements. The results point out that the fibrils still maintain the steric zipper structure after mutating alanine at residue 117 to isoleucine, which has a very bulky side chain.
In addition, we have also investigated the amyloid fibrils formed by the residues 127-147 of the human prion protein (HuPrP127-147, Ac-GYMLGSAMSRPIIHFGSDYED-NH2) by TEM to trace the initial stages of amyloid fibrils formation and observe the morphology of the fibril and spherical aggregates. The fibrils are about 6 nm in width from the sample incubated for 5 minutes only. The distance between the β-strands was determined by ssNMR to be 5±0.1 Å at the proline residue. It demonstrates that the side chain structure of P137 does not disrupt the β-sheet structure.
Subjects
ssNMR
Prion
Amyloid fibril
SDGs
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-99-R97223166-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):55a3bf1aa3a5ec11b3c759d7bc644f18