Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence
- Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010 (Australia)
- Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010 (Australia)
Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP{sup C}) into a disease associated form (PrP{sup Sc}). Recombinant PrP can be refolded into either an {alpha}-helical rich conformation ({alpha}-PrP) resembling PrP{sup C} or a {beta}-sheet rich, protease resistant form similar to PrP{sup Sc}. Here, we generated tetracysteine tagged recombinant PrP, folded this into {alpha}- or {beta}-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished {beta}-PrP from {alpha}-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the {alpha}-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the {beta}-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP{sup Sc} from PrP{sup C}. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.
- OSTI ID:
- 21255927
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 380, Issue 3; Other Information: DOI: 10.1016/j.bbrc.2009.01.120; PII: S0006-291X(09)00165-X; Copyright (c) 2009 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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