. Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH. Nat Chem Biol. 2007 Nov 4;


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-EDT(2) and ReAsH-EDT(2). 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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  1. This exciting study describes a novel method for detecting the misfolding of proteins in live cells using the FlAsH system. By splitting the bipartite tetracysteine motif required for FlAsH fluorescence between distant locations in the amino acid sequence of a protein that are only brought into close proximity by correct folding, the authors are able to use the FlAsH dye to detect whether proteins fold correctly or not, even in live cell culture. This work is of great potential significance to those working on diseases associated with protein misfolding, as the ability to detect misfolding events occurring in situ in live cells has the potential to transform our understanding of the dynamics of such events and how they relate to cellular toxicity. Furthermore, by demonstrating the ability of this system to detect protein-protein interactions, this work offers the tempting possibility of being able to use this approach to study, directly and in detail, protein aggregation phenomena in a cellular environment.

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