Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mammalian cell–based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity


Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells1,2, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins2,3. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to β-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: RRL1 selection for improved tetracysteine sequences.
Figure 2: RRL2 selection and analysis of the optimized flanking residues.
Figure 3: Contrast improvement quantified by flow cytometry.
Figure 4: Fusion of optimized tetracysteines to β-actin.


  1. Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  Google Scholar 

  2. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  Google Scholar 

  3. Stroffekova, K., Proenza, C. & Beam, K.G. The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins. Pflugers Archiv. Eur. J. Physiol. 442, 859–866 (2001).

    Article  CAS  Google Scholar 

  4. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).

    Article  CAS  Google Scholar 

  5. Ju, W. et al. Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors. Nat. Neurosci. 7, 244–253 (2004).

    Article  CAS  Google Scholar 

  6. Marek, K.W. & Davis, G.W. Transgenically encoded protein photoinactivation (FIAsH-FALI): Acute inactivation of synaptotagmin I. Neuron 36, 805–813 (2002).

    Article  CAS  Google Scholar 

  7. Tour, O., Meijer, R.M., Zacharias, D.A., Adams, S.R. & Tsien, R.Y. Genetically targeted chromophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508 (2003).

    Article  CAS  Google Scholar 

  8. Andresen, M., Schmitz-Salue, R. & Jakobs, S. Short tetracysteine tags to β-tubulin demonstrate the significance of small labels for live cell imaging. Mol. Biol. Cell 15, 5616–5622 (2004).

    Article  CAS  Google Scholar 

  9. Hoffmann, C. et al. A FlAsH-based FRET approach to determine G-protein coupled receptor activation in living cells. Nat. Methods 2, 171–176 (2005).

    Article  CAS  Google Scholar 

  10. Panchal, R.G. et al. In vivo oligomerization and raft localization of Ebola virus protein VP40 during vesicular budding. Proc. Natl. Acad. Sci. USA 100, 15936–15941 (2003).

    Article  CAS  Google Scholar 

  11. Rice, M.C., Bruner, M., Czymmek, K. & Kmiec, E.B. In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisae . Mol. Microbiol. 40, 857–868 (2001).

    Article  CAS  Google Scholar 

  12. Rice, M.C., Czymmek, K. & Kmiec, E.B. The potential of nucleic acid repair in functional genomics. Nat. Biotechnol. 19, 321–326 (2001).

    Article  CAS  Google Scholar 

  13. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  14. Chen, I. & Ting, A.Y. Site-specific labeling of proteins with small molecules in live cells. Curr. Opin. Biotechnol. 16, 35–40 (2005).

    Article  CAS  Google Scholar 

  15. Griffin, B.A., Adams, S.R., Jones, J. & Tsien, R.Y. Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol. 327, 565–578 (2000).

    Article  CAS  Google Scholar 

  16. Benhar, I. Biotechnological applications of phage and cell display. Biotechnol. Adv. 19, 1–33 (2001).

    Article  CAS  Google Scholar 

  17. Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    Article  CAS  Google Scholar 

  18. Webb, Y., Hermida-Matsumoto, L. & Resh, M.D. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J. Biol. Chem. 275, 261–270 (2000).

    Article  CAS  Google Scholar 

  19. Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    Article  CAS  Google Scholar 

  20. Daugherty, P.S., Iverson, B.L. & Georgiou, G. Flow cytometric screening of cell-based libraries. J. Immunol. Methods 243, 211–227 (2000).

    Article  CAS  Google Scholar 

  21. Nguyen, A.W. & Daugherty, P.S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat. Biotechnol. 23, 355–360 (2005).

    Article  CAS  Google Scholar 

  22. Zufferey, R., Donello, J.E., Trono, D. & Hope, T.J. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73, 2886–2892 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Cubitt, A.B., Woollenweber, L.A. & Heim, R. Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. Methods Cell Biol. 58, 19–30 (1999).

    Article  CAS  Google Scholar 

Download references


We thank the VA Research Flow Cytometry Core Facility at UCSD and W. Coyt Jackson for flow cytometry assistance, Larry Gross for mass spectrometry support, Paul Steinbach and Qing Xiong for laboratory assistance, Thomas Hope (University of Illinois at Chicago) for the Bluescript SK+ WPRE plasmid, and members of the Tsien laboratory and FlAsHers group for helpful discussions. Some of the work included here was conducted at the National Center for Microscopy and Imaging Research, which is supported by US National Institutes of Health grant RR04050 (to Mark H. Ellisman, University of California, San Diego). This work was supported by NIH NS27177, GM 72033, Department of Energy DE-FG03-01ER63276 and the Howard Hughes Medical Institute.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Roger Y Tsien.

Ethics declarations

Competing interests

R.Y.T. is coinventor on certain patents assigned to the University of California, the value of which could be affected by this publication.

Supplementary information

Supplementary Fig. 1

Analysis of unique sequences isolated in Sort 14. (PDF 116 kb)

Supplementary Fig. 2

Inhibition of tetracysteine-specific membrane localization. (PDF 121 kb)

Supplementary Fig. 3

Dithiol resistance of alanine mutants point to key residues. (PDF 76 kb)

Supplementary Table 1

Quantum yields of FlAsH and ReAsH bound to optimized tetracysteine sequences fused to fluorescent proteins. (PDF 6 kb)

Supplementary Table 2

Oligonucleotide primer sequences. (PDF 30 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Martin, B., Giepmans, B., Adams, S. et al. Mammalian cell–based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat Biotechnol 23, 1308–1314 (2005).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing