NR ATRA
AU Requena,J.R.; Baier,M.; Sajnani,G.; Guitian-Fernandez,E.; Schwarz,A.; Onisko,B.
TI Probing PrPsc structure using chemical cross-linking and mass spectrometry
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Structure of PrP and molecular determinants of infectivity STRCT-16
PT Konferenz-Poster
AB Elucidation of the structure of PrPsc is essential to understand prion infectivity, but has been hampered by the insolubility of PrPsc. We are using a novel approach based on combination of chemical cross-linking, proteolytic digestion, and mass spectrometry (MALDI-TOF and nanoLC-ESI-QqTOF), to gain structural information on PrP 27-30 purified from the brains of scrapie-infected Syrian hamsters. The rationale of the approach is to identify pairs of specific amino acid residues that are close enough to each other so as to react with a bifunctional reagent of a given length. We cross-linked PrP 27-30 with the amino-specific reagent bis(sulfosuccinimidyl) suberate (BS3), obtaining dimers, trimers and larger oligomers that were separated by SDS-PAGE. Digestion and mass spectrometric analysis, showed that BS3 reacted preferentially with Gly90. A cross-link involving two Gly90 amino termini was found in cross-linked PrP27-30 dimers, but not in intra-molecularly cross-linked monomers or control samples. Its identity was further confirmed by MS/MS. This indicates the spatial proximity of Gly90 amino termini in PrP27-30. 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 PrPsc. In ongoing studies, we have also cross-linked PrP 27-30 with the zero-distance cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), which catalyzes transformation of salt bridges into peptide bonds. Formation of dimers, trimers, etc., indicates the presence of such contacts between subunits of the PrPsc aggregate. Efforts to identify the amino acid residues involved are under way.
AD Jesús R. Requena, Gustavo Sajnani, Esteban Guitian-Fernández, University of Santiago de Compostela, Galiza, Spain; Michael Baier, Anja Schwarz, Robert Koch Institute, Berlin, Germany; Bruce Onisko, Western Regional Research Center, USDA, Albany, CA, USA
SP englisch
PO Deutschland