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Tetracysteine‐tagged prion protein allows discrimination between the native and converted forms
Author(s) -
Gašperšič Jernej,
HafnerBratkovič Iva,
Stephan Michel,
Veranič Peter,
Benčina Mojca,
Vorberg Ina,
Jerala Roman
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2010.07619.x
Subject(s) - prion protein , chemistry , monomer , biophysics , helix (gastropod) , transfection , fluorescence , amyloid (mycology) , reagent , biochemistry , biology , gene , medicine , inorganic chemistry , ecology , physics , disease , organic chemistry , pathology , quantum mechanics , snail , polymer
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 fibrilized form of TC‐tagged PrP, and showed that TC‐tagged mPrP could be detected on transfected cells, thereby expanding the potential use of this method for the detection and study of conformational diseases. Structured digital abstract• MINT‐7709757 : Prp (uniprotkb: P04925 ) and Prp (uniprotkb: P04925 ) bind ( MI:0407 ) by electron microscopy ( MI:0040 ) • MINT‐7709744 : Prp (uniprotkb: P04925 ) and Prp (uniprotkb: P04925 ) bind ( MI:0407 ) by circular dichroism ( MI:0016 ) • MINT‐7709730 : Prp (uniprotkb: P04925 ) and Prp (uniprotkb: P04925 ) bind ( MI:0407 ) by fluorescence technology ( MI:0051 )