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Title: PROBING PRPSC STRUCTURE USING CHEMICAL CROSS-LINKING AND MASS SPECTROMETRY: EVIDENCE OF THE PROXIMITY OF GLY90 AMINO TERMINAL IN THE PRP 27-30 AGGREGATE

Author
item Onisko, Bruce
item FERNANDEZ, ESTEBAN - UNIVERSITY OF SANTIAGO
item FREIRE, MARIA - ROBERT KOCH INSTITUTE
item SCHWARZ, ANJA - ROBERT KOCH INSTITUTE
item BAIER, MICHAEL - ROBERT KOCH INSTITUTE
item CAMINA, FELIX - UNIVERSITY OF SANTIAGO
item GARCIA, JAVIER - UNIVERSITY OF SANTIAGO
item VILLAMARIN, RODRIGUEZ-SEGAD - UNIVERSITY OF SANTIAGO
item REQUENA, JESUS - UNIVERSITY OF SANTIAGO

Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/25/2005
Publication Date: 7/7/2005
Citation: Onisko, B.C., Fernandez, E.G., Freire, M.L., Schwarz, A., Baier, M., Camina, F., Garcia, J.R., Villamarin, R.S., Requena, J.R. 2005. Probing prpsc structure using chemical cross-linking and mass spectrometry: evidence of the proximity of gly90 amino terminal in the prp 27-30 aggregate. Biochemistry. 44(30):10100-10109

Interpretive Summary: Prions are proteins that are responsible for development of fatal brain diseases such as BSE (mad cow disease). This and related diseases are spread by consumption of contaminated animal products. It is the unusual structure of prions which make them dangerous. However, the precise structure of prions is unknown. In order to better understand the shape of prions, scientists in the Foodborne Contaminants Research Unit, Western Regional Research Center, Albany, CA undertook structural studies of prion proteins. For the first time, chemical cross linking agents were used to measure the precise distances between specific amino acids of the prion protein. The results have proven useful to other scientists who have made computer models to attempt to understand prion structure. Detailed knowledge of the structure of prions will allow a basis to establish a science-based regulatory policy to ensure a safe food supply.

Technical Abstract: 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 is (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).