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UCSB Team Joins Flow Cytometry, FRET to Describe Protein Interactions in Live Cells

A technique that combines flow cytometry with genetically encoded fluorescent proteins may provide scientists with a new, higher throughput way to identify and characterize protein-protein interactions in living cells, according to recently published research out of the University of California, Santa Barbara.
The method, which can be applied to a variety of eukaryotic cell types, may also eventually be useful in a wide array of proteomic, metabolomic, and drug-discovery applications. In addition, the technique is an example of the increasingly varied uses of flow cytometry in biological research and drug discovery.
The research, which is described in the Dec. 5 issue of the Proceedings of the National Academy of Sciences, builds on earlier work by two of the PNAS paper’s co-authors: Patrick Daugherty, an assistant professor of chemical engineering at UCSB; and graduate student Annalee Nguyen.
Specifically, Nguyen and Daugherty developed a pair of fluorescent proteins based on the widely used cyan and yellow fluorescent proteins but optimized for performing Förster resonance energy transfer (FRET) studies of protein-protein interactions in living cells – work that was published last year in Nature Biotechnology.
The new proteins, dubbed CyPet and YPet, were developed via a series of mutagenesis techniques, and feature a substantially expanded dynamic FRET range. Typically in FRET, a cellular molecule is expressed along with a donor fluorophore whose emission overlaps the excitation of an acceptor fluorophore attached to another cellular molecule
Researchers can tell if the tagged molecules are very close to one another (less than 10 nm apart) and presumably interacting by exciting the donor and seeing if it transfers its energy to the acceptor. However, while many synthetically designed FRET tags exhibit good sensitivity and dynamic range, tags based on the GFP family of proteins do not perform as well – which is why the UCSB scientists developed their improved versions.
In the Nature Biotechnology, paper, Daugherty and Nguyen described how the improved FRET pair could be used with flow cytometry to conduct high-throughput screening of caspase-3-dependent apoptotic cells. This time, as described in the PNAS paper, the researchers focused their attention on the burgeoning problem of mapping protein interactions in cells, or the interactome.
The yeast two-hybrid (Y2H) system has been the primary genetic tool used to discover potential interaction partners for proteins. And although it has been used to great effect in shaping the interactome of yeast, fruitflies, and nematodes, “only a small fraction of an estimated 105 to 106 human-proteome interactions have been unambiguously identified, primarily by using Y2H and mass spectrometry approaches,” the researchers wrote. to 10 human-proteome interactions have been unambiguously identified, primarily by using Y2H and mass spectrometry approaches,” the researchers wrote.
“Unfortunately, high-throughput Y2H assays are typically prone to a high frequency of false positives that complicate the interpretation of interaction data,” the researchers wrote. In addition, this approach is not ideal for “investigating the real-time dynamics in protein networks essential for understanding cell function because detection … is based on a non-reversible process,” they added.
Daugherty and Nguyen, along with collaborators from the Bauer Center for Genomics Research at Harvard University, decided to apply the flow cytometric FRET technique to directly detect and screen peptide libraries for protein interactions in a variety of living cells. In all cases, they used a BD Biosciences FACSAria flow cytometer for analysis.
First, they provided proof of principle by analyzing well-known interaction partners – a specific domain from monocytic adaptor protein, or Mona, and a high-affinity peptide ligand called P2. They expressed Mona with the YPet fluorophore, tethered P2 to CyPet, and expressed the hybrid proteins in E. coli cells. Flow cytometric analysis revealed that FRET was apparent in only cells expressing both hybrid proteins, and not in cells that contained only one or neither of the fluorescent tags.
Next, they screened libraries of peptide ligands labeled with CyPet against cells expressing the Mona protein tagged with YPet. They gradually enriched for cells demonstrating a strong FRET signal by repeated flow cytometric analysis, and then determined the relative strengths of the protein-protein interactions by analyzing the FRET signals more closely in cell lysates.

“Considering that fluorescent proteins are functional in a variety of cell types and compartments, FRET hybrids should be extendable to a broad range of host cells.”

Not only were they able to identify several ligands of Mona, they were able to rank them according to their affinity, and were able to show that most of them shared similar binding motifs in their sequences. Furthermore, “all but one of the Mona ligands identified by FRET hybrids possessed a motif known to be physiologically relevant,” the researchers wrote.
Lastly, they repeated the experiments using yeast cells and human embryonic kidney cells, and actually saw markedly improved FRET signal dynamic ranges, suggesting that the technique may be more readily applied in these more physiologically relevant cell types. The reason they chose E. coli to work with in the first place was because they are easier to grow, manipulate, and genetically transform.
“Considering that fluorescent proteins are functional in a variety of cell types and compartments, FRET hybrids should be extendable to a broad range of host cells,” the researchers wrote. “The ability to screen intracellular protein interactions within a more diverse group of cell types will enable screening in the environment that is most relevant to the interaction of interest.”
It is unclear whether the researchers will continue to focus heavily on using their technique to expand the map of human protein-protein interactions, or whether they will consider using the method to explore other life sciences applications. Calls and e-mails to the authors were not returned in time for this publication.
However, one can easily make the leap from a high-throughput method for characterizing protein-protein interactions to one for screening small molecules that disrupt those same reactions. And, in their original Nature Biotechnology paper describing the optimized FRET pairs, Daugherty and Nguyen wrote, “CyPet-YPet enables utilization of standard flow cytometry instrumentation for high-throughput analysis and screening applications in signal transduction, protein interactions, and enzyme engineering.

“The ability to perform a wider variety of non-invasive FRET measurements in living cells should be particularly useful in proteomics, metabolic and cellular engineering, and drug discovery,” they added.

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