Title
Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence
Author
Bradley M. Coleman, Rebecca M. Nisbet, Sen Han, Roberto Cappai, Danny M. Hatters, Andrew F. Hill
Year
2009
Journal
Biochemical and Biophysical Research Communications
Abstract
Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrPC) into a disease associated form (PrPSc). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrPC or a β-sheet rich, protease resistant form similar to PrPSc. Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91–111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-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 PrPSc from PrPC. 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.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Protein folding, Biochemistry