title: Progress towards structural understanding of infectious sheep PrP-amyloid.
abstract: 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 α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct β-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 β-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.
authors.affiliation: a Institute of Complex Systems; ICS-6: Structural Biochemistry; Forschungszentrum Jülich (FZJ) ; Jülich , Germany.
authors.forename: Henrik
authors.initials: H
authors.lastname: Müller
authors.forename: Oleksandr
authors.initials: O
authors.lastname: Brener
authors.forename: Olivier
authors.initials: O
authors.lastname: Andreoletti
authors.forename: Timo
authors.initials: T
authors.lastname: Piechatzek
authors.forename: Dieter
authors.initials: D
authors.lastname: Willbold
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Legname
authors.forename: Henrike
authors.initials: H
authors.lastname: Heise
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D018625:Microscopy, Atomic Force; D011328:Prions / Q000737:chemistry* / Q000472:pathogenicity; D011487:Protein Conformation; D012756:Sheep
chemical_list: D000682:Amyloid; D011328:Prions
keywords: amyloid fibril; atomic force microscopy; infectious diseases; neurodegenerative diseases; protein structure; solid-state NMR spectroscopy