Probing the structure of the infectious amyloid form of the prion-forming domain of HET-s using high resolution hydrogen/deuterium exchange monitored by mass spectrometry

J Biol Chem. 2005 Apr 8;280(14):13220-8. doi: 10.1074/jbc.M413185200. Epub 2005 Jan 12.

Abstract

The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218-289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218-289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218-289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218-289) sequence. The HET-s-(218-289) fibrils are thus likely to be organized as a succession of beta-sheet segments interrupted by short turns or short loops.

MeSH terms

  • Amino Acid Sequence
  • Amyloid / chemistry
  • Amyloid / metabolism
  • Amyloid / ultrastructure*
  • Deuterium / chemistry*
  • Fungal Proteins / chemistry
  • Fungal Proteins / metabolism
  • Fungal Proteins / ultrastructure*
  • Hydrogen / chemistry*
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / metabolism
  • Podospora / chemistry
  • Prions / chemistry
  • Prions / metabolism
  • Prions / ultrastructure*
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / ultrastructure
  • Spectrometry, Mass, Electrospray Ionization / methods*

Substances

  • Amyloid
  • Fungal Proteins
  • HET-S protein, Podospora anserina
  • Peptides
  • Prions
  • Recombinant Proteins
  • Hydrogen
  • Deuterium