Rather than function in isolation, proteins are part of a larger network of interactions necessary for life. By mapping out protein-protein interactions in biological systems, researchers can obtain a better understanding of health and disease.
However, given the small size of protein molecules and their often-transient binding to one another, scientists have had difficulty directly observing protein-protein interactions. Researchers led by Farid Ghadessy, Group Leader at the p53 Laboratory, A*STAR, have found a way to simplify the detection and observation of protein-protein interactions by reverse-engineering an enzyme from nature.
In collaboration with Robert Robinson at the Institute of Molecular and Cell Biology (IMCB), A*STAR, and colleagues in Thailand, Ghadessy modified CueO, a copper-oxidizing enzyme from the bacterium Escherichia coli, into a protein sensor. “We decided to use CueO because it is stable across a wide range of temperatures, its structure is well known, and the reaction it catalyzes is easily observed,” he explained.
The next step involved Ghadessy’s team grafting fragments of the tumor suppressor protein p53 onto CueO, which allowed them to gain insights into how another protein, MDM2, interacts with p53. “Elevated levels of MDM2 can indicate the presence of certain cancers,” Ghadessy said.
When MDM2 binds to the fragments of p53 engrafted onto CueO, the enzymatic activity of CueO was triggered, resulting in the conversion of a colorless compound into a colored product. Importantly, the color intensity increased proportionately with the concentration of MDM2.
“We discovered that our biosensors were sensitive in the micromolar range, and that we could use them to detect protein-protein interactions in a single test tube with easy visual indication, without the need for purification or washing steps,” said Ghadessy. In addition, the researchers showed that their CueO-based biosensor specifically detects interactions between MDM2 and p53—no color change occurred in the presence of inhibitors that prevent MDM2 from binding to p53.
To further demonstrate the customizability of their CueO-based biosensor, the researchers engrafted an antibody onto CueO, showing that CueO-mediated color change only occurred when the antibody binds to a viral protein. “In the future, we plan to make other biosensors based on the CueO enzyme, and we expect these biosensors to be very helpful in addressing important structural questions about the cancer-related proteins we study,” said Ghadessy.