title: 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.
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.
authors.affiliation: Institut Européen de Chimie et Biologie, CNRS UMR 5144, 2 rue Robert Escarpit, 33600 Pessac, France. nazabal@org.chem.ethz.ch
authors.forename: Alexis
authors.initials: A
authors.lastname: Nazabal
authors.forename: Marie-Lise
authors.initials: ML
authors.lastname: Maddelein
authors.forename: Marc
authors.initials: M
authors.lastname: Bonneu
authors.forename: Sven J
authors.initials: SJ
authors.lastname: Saupe
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Schmitter
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D003903:Deuterium / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D006859:Hydrogen / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048468:Podospora / Q000737:chemistry; D011328:Prions / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000682:Amyloid; D005656:Fungal Proteins; C083891:HET-S protein, Podospora anserina; D010455:Peptides; D011328:Prions; D011994:Recombinant Proteins; D006859:Hydrogen; D003903:Deuterium