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Yale Scientists Use ‘Profluorescent’ Dyes; Biarsenicals Smaller, Less Toxic Than GFPs

Proteins are often tagged using variants of the green fluorescent protein, although they are very large and often toxic to living cells, and are difficult to work with and monitor because they tend to aggregate.
Investigators at Yale University have developed a new technology, dubbed bipartite tetracysteine display, that tags proteins using fluorescence-emitting small molecules. These “profluorescent” biarsenal dyes 4,5-bis (1,3,2-dithiarsolan-2-yl) fluorescein (or FlAsH-EDT2) and 4,5-bis (1,3,2-dithiarsolan-2-yl) resorufin (or ReAsH-EDT2) can easily enter cells and fluoresce when they bind a CCPGCC tag sequence within a recombinant protein in the cytosol, endoplasmic reticulum, and plasma membrane via a thiol-arsenic ligand reaction that converts the nonfluorescent 1,2-ethanedithiol (EDT)-bound forms of FlAsH and ReAsH into highly fluorescent protein bound complexes.
According to the researchers, this marks the first time that these profluorescent dyes have been used to identify interactions among proteins, although they have been used for about a decade to bind single proteins. 
The researchers divided the amino acid tag for the dye into two pieces, and located each piece of the tag far apart on the tag sequences of the recombinant protein that they expressed in living cells. They then monitored cells exposed to the dye. When the protein folded correctly, the two parts of the tag came together and the compound fluoresced.
The investigators published their work in the December issue of Nature Chemical Biology.
The paper describes a proof-of-principle study of the technology, which is still being tested. But if validated by the researchers in ongoing work, the technology could provide a way for researchers to detect diseases in which protein misfolding is a suspected mechanism, such as Alzheimer’s disease, cystic fibrosis, and Parkinson’s disease, the authors say.
In their ongoing validation work, the investigators are using their technology to generate more sensitive sensors for protein-protein interactions and to identify the location at which protein-protein complexes form at high resolution using electromicroscopy.
Senior author Alanna Schepartz, Milton Harris professor of chemistry and the Howard Hughes Medical Institute professor at Yale, told CBA News sister publication ProteoMonitor that she and her team are also using it to identify the trafficking patterns of proteins that assemble in different compartments in the cell.
Schepartz spoke to CBA News last week about the technology and its potential as a tool for drug discovery.

Can you give me a little background on this technology? Is it an alternative to green fluorescent protein tagging?
Not exactly. It’s a way to visualize the formation of protein complexes. I guess it’s sort of an alternative to GFPs. It’s a way to visualize protein complexes and alternative protein conformations in a way that relies on small, cell-permeable dyes and not large fluorescent proteins.
How exactly does this technology work?
The dyes that we use have the ability to bind to cysteine thiols on proteins. Its been known for about a decade that biarsenicals form highly fluorescent complexes when bound to proteins containing four cysteines arranged in a linear motif.
The molecules that we used, which were originally reported by Roger Tsien’s lab at the University of California at San Diego in 1998, have been widely used to simply tag proteins inside both reducing and oxidizing environments of the cell.
It was previously thought that there was very little leeway in the linear sequence of cysteines required to generate a high affinity, bright complex. Our work shows that there can be enormous leeway in that sequence as long as the stretch of protein or amino acids that separates the two pairs of cysteines folds into a structure that brings the thiols together in the right orientation.    
We are excited about this technology for many reasons. For example, you could imagine using this strategy to screen small molecule libraries for members that activate or inactivate membrane-bound receptors.
How exactly would these dyes be used in screening?
It depends on the system. It could be used to identify small molecules that activate or deactivate receptors whose function depends on monomer-dimer equilibrium.
What is the next step in your work?

The paper in Nature Chemical Biology describes a proof of principle. People in the lab are working on applying this idea in several different ways.  

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