|Sajnani, Gustavo - UNV.SANTIAGO, SPAIN|
|Pastrana, Miguel - UNV.SANTIAGO, SPAIN|
|Requena, Jesus - UNV.SANTIAGO, SPAIN|
Submitted to: Journal of Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 24, 2008
Publication Date: July 1, 2008
Citation: Sajnani, G., Pastrana, M.A., Dynin, I.A., Onisko, B.C., Requena, J.R. 2008. Insights on scrapie prion protein (prpsc) structure obtained by limited proteolysis and mass spectrometry. Journal of Molecular Biology. 382(2008):88-98. Interpretive Summary: Prions were treated with proteases to see which places got cleaved the easiest. From this data a molecular model of what a prion might look like was developed."
Technical Abstract: Elucidation of the structure of 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 if contained in loops, and hardly cleave '-sheet stretches. We treated PrPSc isolated from brains of hamsters infected with 263K and Drowsy prions with different concentrations of proteinase K (PK). After PK deactivation, PrPSc was denatured, reduced, and cleaved at Cys179 with 2-nitro-5-thiocyanatonitrobenzoic acid (NTCB). Fragments were analyzed by nanoHPLC-MS and MALDI. Besides the known cleavages at positions 90, 86, 92 for 263K and 86, 90, 92, 98 and 101 for Dy, our data clearly demonstrate the existence of additional cleavage sites at more internal positions, including 117, 119, 135, 139 142 and 151 in both strains.