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US-Swiss Team Develops Non-Toxic Alternative to Fluorescence Tags

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While fluorescence-based technologies have allowed proteomics researchers to analyze protein-protein interactions, a major drawback has been their disruptive nature to live cells.
 
But now a team of researchers from Yale University and the University of Zurich has developed a technology they say can allow scientists to analyze protein activity in living cells.
 
In a paper published in the current issue of Nature Chemical Biology, available here, the researchers say their technology, dubbed bipartite tetracysteine display, is less disruptive to protein functions than tagging methods using variations of green fluorescent protein, and “is also compatible with functional assays that detect protein misfolding and aggregation in bacteria.”
 
The paper is a proof-of-principle study of the technology, which is still being tested. But if validated by the researchers in ongoing work, it 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.
 
Don't FRET
  
According to the research team, fluorescence has “revolutionized” cell biology, and methodologies based on green fluorescent protein and fluorescent protein variants, in particular Forster resonance energy transfer, or FRET, have allowed researchers to probe dynamic and complex processes such as protein association, conformational changes, and post-translational modifications.
 
However, proteins that are tagged using such technologies are very large, often toxic to living cells, and can aggregate, making them difficult to handle and monitor.
 
What the researchers set out to do was “identify a small-molecule-based method that would allow one to selectively image alternative conformations or assembly states,” Alanna Schepartz, a professor of chemistry at Yale and the senior author of the paper, told ProteoMonitor last week.
 
The approach she and her colleagues chose is based on two molecules that have been used as protein tags before: “profluorescent” biarsenical dyes 4,5-bis (1,3,2-dithiarsolan-2-yl fluorescein, also known as FlAsH-EDT2, and 4,5-bis (1,3,2-dithiarsolan-2-yl) resorufin, also known as ReAsH-EDT2.
 
“It’s been known for a decade that the biarsenicals that our study is based upon can be used to recognize proteins that contain a linear arrangement of four cysteines,” Schepartz said. “What we did was ask … whether those four cysteines needed to be in a linear arrangement, or could they, in fact, be presented by regions of a protein that are very far apart in primary sequence, but brought in proximity by virtue of a protein folding event, or a conformational change, or by virtue of formation of a protein-protein interaction?”
 
In addition, the study shows that FlAsH and ReAsHcan differentiate between alternative folding states “for which there were no good methods that offer the benefit of temporal control,” Schepartz said.
 
Both FlAsH and ReAsH are known to be able to easily enter cells and become fluorescent when they bind to a specific amino acid tag sequence within a protein.
 
“It is well established that addition or deletion of even a single amino acid to or from the optimized Pro-Gly sequence separating the two cysteine pairs significantly destabilizes the resulting biarsenical complexes (by up to 23,000-fold) and decreases their quantum yield (up to 500 percent),” the authors say in the paper. This suggested to them that the tetracysteine motif could be used to identify protein conformation of protein-protein binding when displayed in a bipartite mode, they say.
 
They chose four structurally characterized polypeptides and protein domains “to evaluate whether the Pro-Gly sequence within the linear tetracysteine motif could be replaced with one or more folded proteins while maintaining biarsenical affinity and fluorescence intensity.” To evaluate intermolecular dipartite tetracysteine display, the researchers used protein-protein dimerization domains from the basic region leucine zipper proteins GCN4 and Jun, modified to contain a single dicysteine motif.
 

“It is conceivable, although we have not demonstrated [it], that one can use this tool to differentiate between different alternatively folded states of a protein. It also in theory could be used to screen libraries of small molecules on the basis of their ability to inhibit or promote a protein-protein interaction.”

To quantify the apparent affinity of each construct for FlAsH and ReAsH, and to determine the relative brightness of each resulting complexes, fluorescence titrations were performed. The researchers found that the relative brightness of the FlAsH complexes of Jun, aPP and GCN4 at saturation are “comparable to that of the optimized linear tetracysteine complex,” they write.
 
Additionally, the apparent affinity of each polypeptide for ReAsH was similar to its affinity for FlAsH, although the resulting ReAsH complexes had lower relative fluorescence intensities, which are in line with the three-fold lower quantum yields reported for ReAsH complexes relative to analogous FlAsH-containing complexes.
 
The in vitro experiments, the researchers write, verify that well-folded polypeptides and protein-protein binding domains, when modified for bipartite tetracysteine display “bind biarsenicals with high affinity and can form complexes with fluorescence intensities that rival the optimized tetracysteine motif,” the authors say.
 
In subsequent steps, they also found that FlAsH binds aPP, Zip4, GCN4, and Jun to form complexes whose secondary structures are similar to the wild-type polypeptide or protein domain.
 
Their study also looked at whether bipartite tetracysteine display can differentiate folded polypeptides from misfolded ones in vitro; the ability of bipartite tetrcysteine display to detect intermolecular protein assemblies in vitro; and whether bipartite FlAsH and ReAsH display can detect protein folding and assembly in live mammalian cells.
 
In conclusion, they found that their method could detect discrete conformational states and stable protein-protein interactions in live cells. In the case of their study, the method was able to distinguish between 40 kiloDalton fusion proteins differing by a single amino acid in the hydrophobic core of aPP or within the dimerization interface of GCN4.
 
In ongoing validation work, Schepartz and her team 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.
 
They also are using it to identify the trafficking patterns of proteins that assemble in different compartments in the cell, Schepartz said.
 
David Morse, a professor of cell biology at the University of Montreal, told ProteoMonitor that the proposed method would work best for proteins where the structure is known and the probe can be placed where it’s likely to succeed when the two protein molecules come together.
 
Because many protein structures are known, the method, he said, potentially could have wide applications. It offers another method for examining an area of research where a lot of work is being conducted, and in conjunction with other methods of analyzing protein-protein interactions, could reveal a more complete picture of what is happening during such interactions, Morse said.
 
Schepartz said that in addition to Alzheimers and Parkinson’s diseases, the method can be used for any ailment in which protein misfolding is suspected.
 

“It is conceivable, although we have not demonstrated [it], that one can use this tool to differentiate between different alternatively folded states of a protein,” Schepartz said. “It also in theory could be used to screen libraries of small molecules on the basis of their ability to inhibit or promote a protein-protein interaction.”

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