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Fluorescent-Three-Hybrid Screens Highlight Protein Interactions

Sarah C.P. Williams

A new approach lets scientists visualize protein-protein interactions at any site in a mammalian cell, something that can’t be done using classic yeast-two-hybrid screens. Learn more...

To study the binding between two proteins—whether they bind, when they bind, and what factors influence their binding—researchers typically turn to yeast-two-hybrid systems.

Fluorescent-three-hybrid study showing the effect of a drug that blocks p53 from binding HDM2

In these screens, a reporter gene is turned on when the proteins of interest attach to one another. “Yeast two-hybrid is obviously a good method for high-throughput screens, but it has tons of limitations,” said Cristina Cordoso, a molecular biologist at Technische Universität Darmstadt in Germany. In particular, she noted, the proteins must be inside the nuclei of yeast or Escherichia coli for the screen to work.

The need for a better way to study protein-protein interactions led Cordoso and her colleagues to develop a new technique, described in the October 24, 2013 issue of Nature Communications. The approach begins with the selection of a localization protein—a protein known to reside in one particular compartment in the cell where researchers would like to study the interaction between two other proteins.

The localization protein is covalently linked to a molecule that binds green fluorescent protein (GFP). One of the proteins of interest is then attached to GFP and the other to a red fluorescent protein (RFP). The GFP-tagged protein is recruited to the chosen location by the GFP-binder on the localization protein. By observing RFP, researchers can determine whether the RFP-tagged protein binds the GFP-tagged one.

“We can measure not only binding but we can also measure disruption of the binding,” pointed out Cardoso. “I don’t know of any other single method that can do it all.”

The new method, dubbed fluorescent-three-hybrid, offers more flexibility than other protein binding screens in terms of the types of proteins that can be studied as well as their localization within the cell. It also can measure the disruption of protein binding with precise timing, suggesting a new way to screen drugs that aim to block protein binding.

To test the new fluorescent-three-hybrid approach, Cardoso’s team observed the interaction between p53 and HDM2, proteins relevant to many cancers. With HDM2 anchored in place, p53 clearly localized to the same spot, indicating binding. When the researchers added drugs known to disrupt that binding, the red p53 quickly dispersed throughout the cell, no longer associating with the anchored HDM2.

“We were very happy with the results,” Cardoso said. “I think this could not have worked much better. It worked in any place we used it, even the cytosol.”

The strength of the technique, she said, lies in the flexibility of the location of the protein screen within living human cells. The ease with which the method can measure the disruption of binding suggests that it could easily be adapted for drug screens. In the current study, for example, Cardoso’s team tested three different drugs designed to disrupt p53-HDM2 binding, observing which drug disrupted binding the fastest and most completely.

Cardoso’s lab is working to make the method more quantitative. They are also fine tuning techniques to control the activation of peptides that block protein binding, with plans to combine those methods with fluorescence-three-hybrid screening to reveal even more details of protein binding and dissociation kinetics.


Herce, H.D., Deng, W., Helma, J., Leonhardt, H., Cardoso, M.C. (2013) Visualization and targeted disruption of protein interactions in living cells. Nature Communications. Published online October 24, 2013.

Keywords:  protein interactions