Fluorescence resonance energy transfer (FRET) is a technique wherein two light-sensitive molecules transfer energy from a donor molecule to an acceptor molecule. The result is emittance of a fluorescent signal by the acceptor molecule which can then be detected.
Credit: Caleb Foster/Shutterstock.com
FRET has many applications in biology. It is widely used in the study of enzyme reaction kinetics and as a detection system in enzyme assays. It has also been used to study protein-protein interactions.
Characterizing protein-protein interactions in living cells provides insight into the overall system of the organism. The yeast two-hybrid (Y2H) system, originally developed in 1989, is the traditional method for studying protein-protein interactions.
Yeast two-hybrid (Y2H) methodology
The Y2H method is based on protein interactions in yeast. Proteins in the system are designated bait and prey. Interaction between the two proteins activates reporter genes that enable growth on a certain medium or a color change.
Y2H systems have been automated for high-throughput studies. They far exceed the capabilities of earlier interaction screens, which cannot detect interactions in vivo.
The use of Y2H assays was notably used to construct the first large-scale protein interaction network in yeast. Similar studies have been carried out in other organisms.
However, Y2H assays have a high rate of false positives that make data interpretation difficult. In some studies, the rate of false positives has been more than 50 percent. Y2H is also not well suited to capture real-time dynamics in protein networks.
As an alternative, FRET has been adapted for protein interaction studies. For those studies, fluorescent proteins that exhibit FRET are genetically encoded in the host organism. These interactions can then be detected in a variety of cell types.
Using FRET for protein interaction studies overcomes some of the disadvantages of the older Y2H method, such as false positives.
All of the protein interaction networks in an organism are known as the interactome. Understanding of the interactome is crucial for defining pathways and finding effective therapies for diseases.
Studying a whole interactome requires more sophisticated methods than the study of individual protein interactions. Y2H is advantageous for this as it is simple, established, and cost-effective. It can be used in large-scale screening as well as smaller protein interaction studies. It can also be carried out in vivo.
However, the use of yeast as host means that interactions from other organisms might not be detected. This could be due to poor expression or a mismatch of post-translational modification, or a lack of binding factors.
Both proteins must be able to access the nucleus, so proteins such as membrane-bound proteins that are not free to enter the nucleus cannot be studied properly.
Overexpression of proteins in Y2H systems can lead to non-specific interactions and a high false-positive rate. Indirect readout is another drawback of the method.
FRET has some advantages for interactome studies. Its ability to monitor protein-protein interactions in real time enables measurement of even the most transient of interactions.
It can also be used in live cells and permits identification of interaction sites. Because fluorophore interactions are reversible, complex interaction dynamics, such as equilibrium dynamics, can be monitored.
The limitation of FRET is that appropriate fluorophores need to be fused to proteins. A strong readout requires close spatial proximity of fluorophores for the energy transfer.
FRET also has lower sensitivity compared to some other fluorescence-based methods due to background auto-fluorescence. It thus requires control experiments to quantify fluorescence intensity.
The process of FRET, based on cells transfected with plasmids encoding fluorescent tagged proteins. CFP and YFP are used as donor and acceptor, respectively. Credit: leogervasio/Youtube.com