Conformational Detection of Prion Protein With Biarsenical Labeling and FlAsH Fluorescence

Biochem Biophys Res Commun. 2009 Mar 13;380(3):564-8. doi: 10.1016/j.bbrc.2009.01.120. Epub 2009 Jan 25.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alzheimer Disease / diagnosis
  • Animals
  • Cysteine / chemistry
  • Fluoresceins / chemistry*
  • Fluorescence
  • Huntington Disease / diagnosis
  • Luminescent Measurements
  • Mice
  • Neurodegenerative Diseases / diagnosis*
  • Organometallic Compounds / chemistry*
  • Parkinson Disease / diagnosis
  • PrPC Proteins / chemistry*
  • Prion Diseases / diagnosis*
  • Protein Folding
  • Protein Structure, Secondary
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry

Substances

  • 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein
  • Fluoresceins
  • Organometallic Compounds
  • PrPC Proteins
  • Recombinant Proteins
  • Cysteine