Conformational transition occurring upon amyloid aggregation of the HET-s prion protein of Podospora anserina analyzed by hydrogen/deuterium exchange and mass spectrometry.
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.
Nazabal
Alexis
A
CNRS FRE 2247 Institut Européen de Chimie et Biologie, 16, Avenue Pey Berland 33607 Pessac, France. a.nazabal@iecb-polytechnique.u-bordeaux.fr
Dos Reis
Suzana
S
Bonneu
Marc
M
Saupe
Sven J
SJ
Schmitter
Jean-Marie
JM
D000682:Amyloid / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D017666:Deuterium Oxide; D005656:Fungal Proteins / Q000737:chemistry*; D006859:Hydrogen; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D010434:Pepsin A / Q000494:pharmacology; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D011328:Prions / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020037:Sordariales / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000682:Amyloid; D005656:Fungal Proteins; C083891:HET-S protein, Podospora anserina; D010455:Peptides; D011328:Prions; D006859:Hydrogen; D003903:Deuterium; D010434:Pepsin A; D017666:Deuterium Oxide