Scrapie Prion Protein Structural Constraints Obtained by Limited Proteolysis and Mass Spectrometry
- 26 September 2008
- journal article
- research article
- Published by Elsevier BV in Journal of Molecular Biology
- Vol. 382 (1), 88-98
- https://doi.org/10.1016/j.jmb.2008.06.070
Abstract
Elucidation of the structure of scrapie prion protein (PrPSc), essential to understand the molecular mechanism of prion transmission, continues to be one of the major challenges in prion research and is hampered by the insolubility and polymeric character of PrPSc. Limited proteolysis is a useful tool to obtain insight on structural features of proteins: proteolytic enzymes cleave proteins more readily at exposed sites, preferentially within loops, and rarely in β-strands. We treated PrPSc isolated from brains of hamsters infected with 263K and drowsy prions with varying concentrations of proteinase K (PK). After PK deactivation, PrPSc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatobenzoic acid. Fragments were analyzed by nano-HPLC/mass spectrometry and matrix-assisted laser desorption/ionization. Besides the known cleavages at positions 90, 86, and 92 for 263K prions and at positions 86, 90, 92, 98, and 101 for drowsy prions, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139, 142, and 154 in both strains. PK concentration dependence analysis and limited proteolysis after partial unfolding of PrPSc confirmed that only the mentioned cleavage sites at the N-terminal side of the PrPSc are susceptible to PK. Our results indicate that besides the “classic” amino-terminal PK cleavage points, PrPSc contains, in its middle core, regions that show some degree of susceptibility to proteases and must therefore correspond to subdomains with some degree of structural flexibility, interspersed with stretches of amino acids of high resistance to proteases. These results are compatible with a structure consisting of short β-sheet stretches connected by loops and turns.Keywords
Funding Information
- Ministerio de Ciencia e Innovación (FP6 2004 FOOD 3B 023183)
- European Commission
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