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

Nature Biotechnology volume 23, pages 1308–1314 (2005)Cite this article

Abstract

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.

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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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr., La Jolla, 92093-0647, California, USA

    Brent R Martin, Ben N G Giepmans, Stephen R Adams & Roger Y Tsien

  2. Biomedical Sciences Graduate Program, University of California, San Diego, 9500 Gilman Dr., La Jolla, 92093-0647, California, USA

    Brent R Martin

  3. Department of Neurosciences, University of California, San Diego, 9500 Gilman Dr., La Jolla, 92093-0647, California, USA

    Ben N G Giepmans

  4. National Center for Microscopy and Imaging Research, University of California, San Diego, 9500 Gilman Dr., La Jolla, 92093-0647, California, USA

    Ben N G Giepmans

  5. Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, 92093-0647, California, USA

    Roger Y Tsien

  6. Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Dr., La Jolla, 92093-0647, California, USA

    Roger Y Tsien

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  1. Brent R Martin
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Correspondence to Roger Y Tsien.

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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)

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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). https://doi.org/10.1038/nbt1136

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