Chemical biology: Smaller can be better

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Researchers have split the FlAsH–ReAsH tetracysteine recognition sequence into two pieces to show that these small-molecule fluorophores can report protein folding or protein–protein interactions.

Fluorescence is not only beautiful, but also useful. Fluorescence imaging tools have become indispensable for cell biology. An entire rainbow of fluorescent proteins is now available to researchers for a multitude of applications. Small-molecule fluorophores, which lack the advantage of genetic encodability but have the advantage of petiteness, are also beneficial in many situations.

Alanna Schepartz of Yale University and her colleagues report the design of a new concept for fluorescent reporting based on the binding of FlAsH or ReAsH biarsenical reagents to a split tetracysteine motif, which they call "bipartite tetracysteine display." These fluoro-phores had been shown several years ago by Roger Tsien and coworkers to specifically label proteins tagged with a tetracysteine (CCPGCC) sequence and only become fluorescent upon forming a biarsenical tetracysteine complex. The bipartite tetracysteine display method now uses these popular fluorophores to report on protein folding or protein-protein interactions. By placing two cysteine-cysteine pairs such that they are far apart in primary sequence—but such that they come into close proximity when the protein is folded (or when two proteins interact)—the fluorescent biarsenical complex can be reconstituted.

The idea came to Schepartz while she was preparing a lecture about FlAsH labeling for her chemical biology class. She was chatting with her postdoc Nathan Luedtke (now a faculty member at the University of Zurich) about her lecture and mentioned that she could not believe that FlAsH (and ReAsH) would only form complexes with the specific CCPGCC sequence. "And Nathan, who's exceptionally quick on the uptake, realized immediately that it could be a really interesting project," recounts Schepartz.

FRET, fluorescence resonance energy transfer, is used to monitor protein folding and protein-protein interactions. Bipartite tetracysteine display offers an alternative that does not require two bulky fluorescent proteins, which can interfere with protein folding and function. "In addition," says Schepartz, "the fluorescent changes induced by the binding of a biarsenical should be higher than the changes in fluorescence due to FRET."

By engineering dicysteine motifs into a set of model protein sequences, the researchers tested whether bipartite tetracysteine display could be used to monitor intramolecular protein folding and intermolecular protein-protein dimerization in vitro. In testing a wide variety of different contexts, they found that "in some cases the proteins [modified for bipartite tetracysteine display] are better behaved than in other cases, but in no case yet have we flat-out failed," says Schepartz, suggesting that the steric requirements for FlAsH or ReAsH binding to the bipartite tetracysteine motif are not quite as stringent as one might think.

Figure 1 |

High resolution image and legend

They also showed that they could distinguish well-folded and misfolded proteins. Proteins with destabilizing point mutations still formed complexes with FlAsH or ReAsH, but the fluorescence was much dimmer in comparison to well-folded proteins (Fig. 1). They have not observed the formation of nonspecific fluorescent complexes of proteins that would otherwise not interact, though Schepartz does clarify that they have not extensively tested for this. Notably, the researchers also showed that their method can be used to detect protein folding and protein-protein interactions in live mammalian cells.

Schepartz and her colleagues believe that bipartite tetracysteine display offers a useful small-molecule alternative to FRET for designing new post-translational modification or protein-protein interaction sensors. The method should also be compatible with other techniques such as electron microscopy and could be used in high-throughput screening applications, for example, to identify small molecules that either stabilize or disrupt protein dimerization.

The future for this method indeed looks very 'bright'.

Allison Doerr

References

Luedtke, N.W. et al. Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH. Nat. Chem. Biol. 3, 779–784 (2007). | Article | PubMed |