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Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size |
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| Authors: | Sun Ying Makarava Natallia Lee Cheng-I Laksanalamai Pongpan Robb Frank T Baskakov Ilia V |
| Affiliation: | 1 Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA 2 Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA 3 Department of Biochemistry and Molecular Biology, University of Maryland, 725 West Lombard Street, Baltimore, MD 21201, USA |
| Abstract: | Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-β-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions—perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including αB-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease. |
| Keywords: | PrP, prion protein rPrP, full-length recombinant prion protein PrPC, cellular isoform of the prion protein PrPSc, disease-associated isoform of the prion protein WT, wild type GdnHCl, guanidine hydrochloride sHsp, small heat shock protein sHsp-MB, small heat shock protein from Methanococcoides burtonii sHsp-PF, small heat shock protein from Pyrococcus furiosus EM, electron microscopy AFM, atomic force microscopy CJD, Creutzfeldt-Jakob disease ThT, thioflavin T |
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