Progress towards structural understanding of infectious sheep PrP-amyloid

Prion. 2014;8(5):344-58. doi: 10.4161/19336896.2014.983754.

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.

Keywords: amyloid fibril; atomic force microscopy; infectious diseases; neurodegenerative diseases; protein structure; solid-state NMR spectroscopy.

MeSH terms

  • Amyloid / chemistry*
  • Animals
  • Circular Dichroism
  • Magnetic Resonance Spectroscopy
  • Microscopy, Atomic Force
  • Prions / chemistry*
  • Prions / pathogenicity
  • Protein Conformation
  • Sheep

Substances

  • Amyloid
  • Prions