Surveying Polypeptide and Protein Domain Conformation and Association with FlAsH and ReAsH

Overview

Journal Nat Chem Biol

Specialties Biochemistry
Biology
Chemistry

Date 2007 Nov 6

PMID 17982447

Citations 57

Authors

Nathan W Luedtke

Rachel J Dexter

Daniel B Fried

Alanna Schepartz

Affiliations

Soon will be listed here.

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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References

Schuler B, Lipman E, Steinbach P, Kumke M, Eaton W . Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence. Proc Natl Acad Sci U S A. 2005; 102(8):2754-9. PMC: 549440. DOI: 10.1073/pnas.0408164102. View

Chudakov D, Lukyanov S, Lukyanov K . Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 2005; 23(12):605-13. DOI: 10.1016/j.tibtech.2005.10.005. View

Hoffmann C, Gaietta G, Bunemann M, Adams S, Oberdorff-Maass S, Behr B . A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat Methods. 2005; 2(3):171-6. DOI: 10.1038/nmeth742. View

Adams S, Campbell R, Gross L, Martin B, Walkup G, Yao Y . New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc. 2002; 124(21):6063-76. DOI: 10.1021/ja017687n. View

Ignatova Z, Gierasch L . Inhibition of protein aggregation in vitro and in vivo by a natural osmoprotectant. Proc Natl Acad Sci U S A. 2006; 103(36):13357-61. PMC: 1569168. DOI: 10.1073/pnas.0603772103. View

Watt R, Voss Jr E . Mechanism of quenching of fluorescein by anti-fluorescein IgG antibodies. Immunochemistry. 1977; 14(7):533-51. DOI: 10.1016/0019-2791(77)90308-1. View

Prasher D, Eckenrode V, Ward W, Prendergast F, Cormier M . Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992; 111(2):229-33. DOI: 10.1016/0378-1119(92)90691-h. View

Junius F, ODonoghue S, Nilges M, Weiss A, King G . High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer. J Biol Chem. 1996; 271(23):13663-7. DOI: 10.1074/jbc.271.23.13663. View

Enninga J, Mounier J, Sansonetti P, Tran van Nhieu G . Secretion of type III effectors into host cells in real time. Nat Methods. 2005; 2(12):959-65. DOI: 10.1038/nmeth804. View

10.

Willemse M, Janssen E, de Lange F, Wieringa B, Fransen J . ATP and FRET--a cautionary note. Nat Biotechnol. 2007; 25(2):170-2. DOI: 10.1038/nbt0207-170. View

11.

Shimomura O, Johnson F, Saiga Y . Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol. 1962; 59:223-39. DOI: 10.1002/jcp.1030590302. View

12.

Ignatova Z, Gierasch L . Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. Proc Natl Acad Sci U S A. 2004; 101(2):523-8. PMC: 327180. DOI: 10.1073/pnas.0304533101. View

13.

Andresen M, Schmitz-Salue R, Jakobs S . Short tetracysteine tags to beta-tubulin demonstrate the significance of small labels for live cell imaging. Mol Biol Cell. 2004; 15(12):5616-22. PMC: 532040. DOI: 10.1091/mbc.e04-06-0454. View

14.

OShea E, Klemm J, Kim P, Alber T . X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 1991; 254(5031):539-44. DOI: 10.1126/science.1948029. View

15.

Dyachok O, Isakov Y, Sagetorp J, Tengholm A . Oscillations of cyclic AMP in hormone-stimulated insulin-secreting beta-cells. Nature. 2006; 439(7074):349-52. DOI: 10.1038/nature04410. View

16.

Giepmans B, Adams S, Ellisman M, Tsien R . The fluorescent toolbox for assessing protein location and function. Science. 2006; 312(5771):217-24. DOI: 10.1126/science.1124618. View

17.

Hammarstrom P, Wiseman R, Powers E, Kelly J . Prevention of transthyretin amyloid disease by changing protein misfolding energetics. Science. 2003; 299(5607):713-6. DOI: 10.1126/science.1079589. View

18.

Blundell T, Pitts J, Tickle I, Wood S, Wu C . X-ray analysis (1. 4-A resolution) of avian pancreatic polypeptide: Small globular protein hormone. Proc Natl Acad Sci U S A. 1981; 78(7):4175-9. PMC: 319751. DOI: 10.1073/pnas.78.7.4175. View

19.

Sreerama N, Venyaminov S, Woody R . Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis. Anal Biochem. 2000; 287(2):243-51. DOI: 10.1006/abio.2000.4879. View

20.

Tsien R . The green fluorescent protein. Annu Rev Biochem. 1998; 67:509-44. DOI: 10.1146/annurev.biochem.67.1.509. View