Together We Shine

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Science  23 Nov 2007:
Vol. 318, Issue 5854, pp. 1219
DOI: 10.1126/science.318.5854.1219b

Green fluorescent protein (GFP) and its variants can be used in Förster resonance energy transfer (FRET) experiments (where emission is a function of the distance between donor and acceptor fluorophores) to monitor dynamic processes such as protein folding or association in intact cells. These experiments come with limitations, however, as the changes in fluorescence intensity may be smaller than cell-to-cell variations, and fusing GFP to the target protein may interfere with function. An alternative readout strategy uses the biarsenical reagents FlAsH -EDT2 and ReAsH-EDT2. These reagents selectively label recombinant proteins containing a tetracysteine sequence (CCPGCC), and only the protein-bound forms fluoresce.

Luedtke et al. show that polypeptides containing a split sequence, with the two cysteine pairs separated in linear sequence but close together when folded, can be labeled with FlAsH or ReAsH to give a fluorescent complex. Labeling of a dimer, where the two pairs were contained on different monomers, was also successful. Fluorescence intensity correlated with the stability of either protein folding or dimerization and allowed the detection of protein folding and assembly in live cells. — VV

Nat. Chem. Biol. 3, 10.1038/nchembio.2007.49 (2007).

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