NR AOGY
AU Nazabal,A.; Dos Reis,S.; Bonneu,M.; Saupe,S.J.; Schmitter,J.M.
TI Conformational transition occurring upon amyloid aggregation of the HET-s prion protein of Podospora anserina analyzed by hydrogen/deuterium exchange and mass spectrometry
QU Biochemistry 2003 Jul 29; 42(29): 8852-61
PT journal article
AB The [Het-s] infectious element of the filamentous fungus Podospora anserina corresponds to the prion form of the HET-s protein. HET-s (289 amino acids in length) aggregates into amyloid fibers in vitro. Such fibers obtained in vitro are infectious, indicating that the [Het-s] prion can propagate as a self-perpetuating amyloid aggregate of the HET-s protein. Previous analyses have suggested that only a limited region of the HET-s protein is involved in amyloid formation and prion propagation. To document the conformational transition occurring upon amyloid aggregation of HET-s, we have developed a method involving hydrogen/deuterium exchange monitored by MALDI-MS. In a first step, a peptide mass fingerprint of the protein was obtained, leading to 87% coverage of the HET-s primary structure. Amyloid aggregates of HET-s were obtained, and H/D exchange was monitored on the soluble and on the amyloid form of HET-s. This study revealed that in the soluble form of HET-s, the C-terminal region (spanning from residues 240-289) displays a high solvent accessibility. In sharp contrast, solvent accessibility is drastically reduced in that region in the amyloid form. H/D exchange rates and levels in the N-terminal part of the protein (residues 1-220) are comparable in the soluble and the aggregated state. These results indicate that amyloid aggregation of HET-s involves a conformational transition of the C-terminal part of the protein from a mainly disordered to an aggregated state in which this region is highly protected from hydrogen exchange.
MH Amyloid/chemistry; Deuterium/chemistry; Deuterium Oxide; Fungal Proteins/*chemistry; Hydrogen; Microscopy, Electron; Pepsin A/pharmacology; Peptide Mapping; Peptides/chemistry; Prions/*chemistry; Protein Conformation; Protein Structure, Tertiary; Sordariales/*metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analysis, Mass; Support, Non-U.S. Gov't
AD a.nazabal@iecb-polytechnique.u-bordeaux.fr, CNRS FRE 2247 Institut Europeen de Chimie et Biologie, 16, Avenue Pey Berland 33607 Pessac, France, and Laboratoire de Genetique Moleculaire des Champignons, Institut de Biochimie et Genetique Cellulaires, UMR 5095 CNRS Universite Victor Segalen Bordeaux 2-1, Rue Camille Saint-Saens, 33077 Bordeaux Cedex, France.
SP englisch
PO USA