title: Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence.
abstract: Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP(C)) into a disease associated form (PrP(Sc)). Recombinant PrP can be refolded into either an alpha-helical rich conformation (alpha-PrP) resembling PrP(C) or a beta-sheet rich, protease resistant form similar to PrP(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(Sc) from PrP(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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Coleman
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Nisbet
authors.forename: Sen
authors.initials: S
authors.lastname: Han
authors.forename: Roberto
authors.initials: R
authors.lastname: Cappai
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Hill
mesh_terms: D000544:Alzheimer Disease / Q000175:diagnosis; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry*; D005453:Fluorescence; D006816:Huntington Disease / Q000175:diagnosis; D008163:Luminescent Measurements; D051379:Mice; D019636:Neurodegenerative Diseases / Q000175:diagnosis*; D009942:Organometallic Compounds / Q000737:chemistry*; D010300:Parkinson Disease / Q000175:diagnosis; D018621:PrPC Proteins / Q000737:chemistry*; D017096:Prion Diseases / Q000175:diagnosis*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
chemical_list: C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D005452:Fluoresceins; D009942:Organometallic Compounds; D018621:PrPC Proteins; D011994:Recombinant Proteins; D003545:Cysteine