New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications

J Am Chem Soc. 2002 May 29;124(21):6063-76. doi: 10.1021/ja017687n.


We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants ( approximately 10(-11) M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Anisotropy
  • Cysteine / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Fluoresceins / chemistry*
  • Fluorescence Polarization
  • Fluorescent Dyes / chemistry*
  • Fluorometry
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Organometallic Compounds / chemistry*
  • Recombinant Proteins / analysis
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / isolation & purification
  • Spectrometry, Fluorescence


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