tailieunhanh - Báo cáo khoa học: Tetracysteine-tagged prion protein allows discrimination between the native and converted forms

The conformational conversion of prion protein (PrP) from a native con-formation to the amyloid form is a hallmark of transmissible spongiform encephalopathies. Conversion is usually monitored by fluorescent dyes, which bind generic amyloids and are less suited for living cell imaging. | Tetracysteine-tagged prion protein allows discrimination between the native and converted forms Jernej GaSperSic1 Iva Hafner-Bratkovic1 Michel Stephan2 Peter Veranic3 Mojca Bencina1 Ina Vorberg4 and Roman Jerala1 5 1 Department of Biotechnology National institute of Chemistry Ljubljana Slovenia 2 Department of Organic and MedicinalChemistry National institute of Chemistry Ljubljana Slovenia 3 Faculty of Medicine Institute of CellBiology Ljubljana Slovenia 4 Institute of Virology TU Munich Germany 5 Faculty of Chemistry and ChemicalTechnology University of Ljubljana Slovenia Keywords biarsenical conversion fibril prion tetracysteine Correspondence R. Jerala Department of biotechnology National institute of Chemistry Hajdrihova 19 1000 Ljubljana Slovenia Fax 386 1 476 0300 Tel 386 1 476 0335 E-mail Received 5 October 2009 revised 19 January 2010 accepted 17 February 2010 doi The conformational conversion of prion protein PrP from a native conformation to the amyloid form is a hallmark of transmissible spongiform encephalopathies. Conversion is usually monitored by fluorescent dyes which bind generic amyloids and are less suited for living cell imaging. We report a new method for the synthesis of membrane-permeable and membrane-impermeable biarsenical reagents which are then used to monitor murine PrP mPrP misfolding. We introduced tetracysteine TC tags into three different positions of mPrP which folded into a native-like structure. Whereas mPrPs with a TC tag inserted at the N-terminus or C-terminus supported fibril formation insertion into the helix 2-helix 3 loop inhibited conversion. We devised a quantitative protease-free method to determine the fraction of converted PrP based on the ability of the fluorescein arsenical helix binder reagent to differentiate between the monomeric and fibril-ized form of TC-tagged PrP and showed that TC-tagged mPrP could be detected on transfected cells thereby expanding the .