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

Nature Chemical Biology volume 3, pages 779–784 (2007)Cite this article

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.

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Figure 1: Intra- and intermolecular bipartite tetracysteine display using FlAsH-EDT2 and ReAsH-EDT2.
Figure 2: Equilibrium binding of FlAsH and ReAsH to polypeptides or protein domains containing a bipartite tetracysteine motif.
Figure 3: Effect of FlAsH binding on the secondary structures of wild-type and variant polypeptides and protein domains.
Figure 4: Misfolded polypeptides and their assemblies bind FlAsH and ReAsH with diminished affinities.
Figure 5: Differentiation of folded and misfolded proteins and assemblies in living cells with ReAsH-EDT2.

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Acknowledgements

This work was supported in part by the US National Institutes of Health and in part by a grant to Yale University, in support of A.S., from the Howard Hughes Medical Institute.

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  1. Nathan W Luedtke

    Present address: Present address: Universität Zürich, Organisch-chemisches Institut, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.,

Authors and Affiliations

  1. Department of Chemistry, Yale University, 225 Prospect Street, New Haven, 06520-8107, Connecticut, USA

    Nathan W Luedtke, Rachel J Dexter, Daniel B Fried & Alanna Schepartz

  2. Department of Chemistry and Molecular, Cellular, and Developmental Biology, Yale University, 219 Prospect Street, New Haven, 06520-8107, Connecticut, USA

    Alanna Schepartz

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Contributions

N.W.L. contributed to experimental design, fluorophore synthesis, in vitro binding analyses, CD and manuscript drafting; R.J.D. contributed to experimental design and live cell experiments; D.B.F. contributed to in vitro binding analyses, CD and characterization of polypeptide-FlAsH complexes; A.S. contributed to experimental design and manuscript drafting.

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Correspondence to Alanna Schepartz.

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Supplementary Figures 1–4, Supplementary Tables 1–3, and Supplementary Methods (PDF 694 kb)

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Luedtke, N., Dexter, R., Fried, D. et al. Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH. Nat Chem Biol 3, 779–784 (2007). https://doi.org/10.1038/nchembio.2007.49

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