NR ASQN
AU Nazabal,A.; Maddelein,M.L.; Bonneu,M.; Saupe,S.J.; Schmitter,J.M.
TI 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
QU The Journal of Biological Chemistry 2005 Apr 8; 280(14): 13220-8
PT journal article
AB 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.
MH Amino Acid Sequence; Amyloid/chemistry/metabolism/*ultrastructure; Deuterium/*chemistry; Fungal Proteins/chemistry/metabolism/*ultrastructure; Hydrogen/*chemistry; Molecular Sequence Data; Peptides/chemistry/genetics/metabolism; Podospora/chemistry; Prions/chemistry/metabolism/*ultrastructure; Protein Structure, Tertiary; Recombinant Proteins/chemistry/metabolism/ultrastructure; Spectrometry, Mass, Electrospray Ionization/*methods
AD Institut Europeen de Chimie et Biologie, CNRS UMR 5144, 2 rue Robert Escarpit, 33600 Pessac, France. nazabal@org.chem.ethz.ch
SP englisch
PO USA