Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH

Nat Chem Biol. 2007 Dec;3(12):779-84. doi: 10.1038/nchembio.2007.49. Epub 2007 Nov 4.

Abstract

Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arsenicals / chemistry*
  • Cell Survival
  • Circular Dichroism
  • HeLa Cells
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Organic Chemicals / chemistry
  • Oxazines / chemistry*
  • Peptides / chemistry*
  • Protein Binding
  • Protein Folding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proteins / chemistry*
  • Proteins / genetics
  • Proteins / metabolism

Substances

  • Arsenicals
  • Organic Chemicals
  • Oxazines
  • Peptides
  • Proteins
  • ReAsH-EDT2 compound
  • calcium green

Associated data

  • PDB/1JUN
  • PDB/1LE3
  • PDB/1PPT
  • PDB/2ZTA
  • PubChem-Substance/26738251
  • PubChem-Substance/26738252
  • PubChem-Substance/26738253
  • PubChem-Substance/26738254