Probing PrPSc Structure Using Chemical Cross-Linking and Mass Spectrometry: Evidence of the Proximity of Gly90 Amino Termini in the PrP 27-30 Aggregate

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• 作者：Bruce Onisko ; Esteban Guitiá ; n Ferná ; ndez ; Marí ; a Louro Freire ; Anja Schwarz ; Michael Baier ; Fé ; lix Camiñ ; a ; Javier Rodrí ; guez Garcí ; a ; Santiago Rodrí ; g
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：August 2, 2005
• 年：2005
• 卷：44
• 期：30
• 页码：10100 - 10109
• 全文大小：227K
• 年卷期：v.44,no.30(August 2, 2005)
• ISSN：1520-4995

文摘

Elucidation of the structure of PrP^Sc continues to be one of the most important and difficultchallenges 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 chemicalcross-linking, proteolytic digestion, and mass spectrometry (MALDI-TOF and nanoLC-ESI-QqTOF), inan attempt to gain structural information about PrP 27-30 purified from the brains of Syrian hamstersinfected with scrapie. The rationale of this approach is to identify pairs of specific amino acid residuesthat are close enough to each other to react with a bifunctional reagent of a given chain length. Wecross-linked PrP 27-30 with the amino-specific reagent bis(sulfosuccinimidyl) suberate (BS³), obtainingdimers, trimers, and higher-order oligomers that were separated by SDS-PAGE. In-gel digestion followedby mass spectrometric analysis showed that BS³ reacted preferentially with Gly90. A cross-link involvingtwo 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 aminotermini 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 butnot those adjacent to each other horizontally in the trimeric building block. Our results constitute the firstmeasured distance constraint in PrP^Sc.