Probing PrPSc structure using chemical cross-linking and mass spectrometry: evidence of the proximity of Gly90 amino termini in the PrP 27-30 aggregate.
Elucidation of the structure of PrP(Sc) continues to be one of the most important and difficult challenges in prion research. This task, essential for gaining an understanding of the basis of prion infectivity, has been hampered by the insoluble, aggregated nature of this molecule. We used a combination of chemical cross-linking, proteolytic digestion, and mass spectrometry (MALDI-TOF and nanoLC-ESI-QqTOF), in an attempt to gain structural information about PrP 27-30 purified from the brains of Syrian hamsters infected with scrapie. The rationale of this approach is to identify pairs of specific amino acid residues that are close enough to each other to react with a bifunctional reagent of a given chain length. We cross-linked PrP 27-30 with the amino-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), obtaining dimers, trimers, and higher-order oligomers that were separated by SDS-PAGE. In-gel digestion followed by mass spectrometric analysis showed that BS(3) reacted preferentially with Gly90. A cross-link involving two Gly90 amino termini was found in cross-linked PrP 27-30 dimers, but not in intramolecularly cross-linked monomers or control samples. This observation indicates the spatial proximity of Gly90 amino termini in PrP 27-30 fibrils. The Gly90-Gly90 cross-link is consistent with a recent model of PrP 27-30, based on electron crystallographic data, featuring a fiber composed of stacked trimers of PrP monomers; specifically, it is compatible with cross-linking of monomers stacked vertically along the fiber axis but not those adjacent to each other horizontally in the trimeric building block. Our results constitute the first measured distance constraint in PrP(Sc).
Onisko
Bruce
B
Western Regional Research Center, United States Department of Agriculture, Albany, California 94710, USA.
Fernández
Esteban Guitián
EG
Freire
María Louro
ML
Schwarz
Anja
A
Baier
Michael
M
Camiña
Félix
F
García
Javier Rodríguez
JR
Rodríguez-Segade Villamarín
Santiago
S
Requena
Jesús R
JR
D000595:Amino Acid Sequence; D000818:Animals; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D019286:Endopeptidase K / Q000378:metabolism; D005998:Glycine / Q000737:chemistry*; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D008647:Mesocricetus; D036103:Nanotechnology; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D018622:PrP 27-30 Protein / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D013388:Succinimides; D018622:PrP 27-30 Protein; C035760:bis(sulfosuccinimidyl)suberate; D012697:Serine Endopeptidases; D014357:Trypsin; C036813:lysyl endopeptidase; D019286:Endopeptidase K; D008239:Lysine; D005998:Glycine