Observation of Highly Flexible Residues in Amyloid Fibrils of the HET-s Prion

Siemer, Ansgar B.; Arnold, Alexandre A.; Ritter, Christiane; Westfeld, Thomas; Ernst, Matthias; Riek, Roland; Meier, Beat H.

doi:10.1021/ja063639x

Cited by 133 publications

(173 citation statements)

References 33 publications

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“…In contrast to this rigid core region, the remainder of the protein backbone in huPrP23-144 fibrils shows a high degree of conformational flexibility (see Variable Temperature NMR Studies of Conformational Dynamics in huPrP23-144 Amyloid). The existence of such rigid and dynamic segments has been previously reported for amyloid fibrils formed by other proteins, including ␣-synuclein (21,22) and HET-s (23,31).…”

mentioning

confidence: 55%

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils

Helmus

Surewicz²,

Nadaud

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

183

298

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A C-terminally truncated Y145Stop variant of the human prion protein (huPrP23-144) is associated with a hereditary amyloid disease known as PrP cerebral amyloid angiopathy. Previous studies have shown that recombinant huPrP23-144 can be efficiently converted in vitro to the fibrillar amyloid state, and that residues 138 and 139 play a critical role in the amyloidogenic properties of this protein. Here, we have used magic-angle spinning solid-state NMR spectroscopy to provide high-resolution insight into the protein backbone conformation and dynamics in fibrils formed by 13 C, 15 N-labeled huPrP23-144. Surprisingly, we find that signals from Ϸ100 residues (i.e., Ϸ80% of the sequence) are not detected above approximately ؊20°C in conventional solid-state NMR spectra. Sequential resonance assignments revealed that signals, which are observed, arise exclusively from residues in the region 112-141. These resonances are remarkably narrow, exhibiting average 13 C and 15 N linewidths of Ϸ0.6 and 1 ppm, respectively. Altogether, the present findings indicate the existence of a compact, highly ordered core of huPrP23-144 amyloid encompassing residues 112-141. Analysis of 13 C secondary chemical shifts identified likely ␤-strand segments within this core region, including ␤-strand 130 -139 containing critical residues 138 and 139. In contrast to this relatively rigid, ␤-sheet-rich amyloid core, the remaining residues in huPrP23-144 amyloid fibrils under physiologically relevant conditions are largely unordered, displaying significant conformational dynamics.protein structure ͉ solid-state NMR ͉ protein misfolding ͉ transmissible spongiform encephalopathies

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mentioning

confidence: 55%

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils

Helmus

Surewicz²,

Nadaud

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

183

298

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“…The observation that fibrils are more dispersed between pH 3.3 and 3.9 than at neutral pH suggests that interactions between charged side chains are involved in their aggregation. There are 8 negatively charged residues and one histidine residue (plus 6 histidines in the his-tag) in the HET-s-(218 -289) sequence that could have a pK a value in this range, and all except one are predicted to be on the surface of the ␤-roll or in flexible loops (16,31).…”

Section: Resultsmentioning

confidence: 99%

Mass Analysis by Scanning Transmission Electron Microscopy and Electron Diffraction Validate Predictions of Stacked β-Solenoid Model of HET-s Prion Fibrils

Sen

Baxa

Simon

et al. 2007

Journal of Biological Chemistry

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“…On the other hand, highly flexible protein segments, undergoing motions in the sub-microsecond range, may be discriminated easily from the rigid core by INEPTbased excitation. 21,[38][39][40][41] In contrast to rigid and highly flexible residues though, signals from semi-flexible regions, such as the fuzzy coat of amyloid fibrils, may be completely missing due to intermediate range molecular motion resulting in severe line broadening and/or disappearance of signals. 12,21,38,42 In order to compare the amounts of rigid and mobile protein segments, we recorded 1D-13 C-CP-and 1D- 13 C-INEPT-NMR spectra.…”

Section: Secondary and Ultrastructural Propertiesmentioning

confidence: 99%

Progress towards structural understanding of infectious sheep PrP-amyloid

Müller

Brener

Andréoletti

et al. 2014

Prion

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The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of a-helical secondary structure in the middle part between »115 to »155, and a distinct b-sheet core C-terminal of residue »155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the b-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.

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Observation of Highly Flexible Residues in Amyloid Fibrils of the HET-s Prion

Cited by 133 publications

References 33 publications

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils

Mass Analysis by Scanning Transmission Electron Microscopy and Electron Diffraction Validate Predictions of Stacked β-Solenoid Model of HET-s Prion Fibrils

Progress towards structural understanding of infectious sheep PrP-amyloid

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