- Förster Resonance Energy Transfer (FRET) is a highly sensitive and non-invasive technique used to study molecular interactions, particularly protein–protein interactions, within living cells. FRET is based on the principle of distance-dependent energy transfer between two fluorophores: a donor and an acceptor. When the donor fluorophore is excited by an appropriate wavelength of light, it can transfer energy non-radiatively to an adjacent acceptor fluorophore, provided they are in close proximity—typically within 1 to 10 nanometers. This energy transfer does not involve the emission of a photon by the donor; instead, the energy is directly transferred through dipole–dipole coupling. Because the efficiency of FRET is inversely proportional to the sixth power of the distance between donor and acceptor, the technique is exquisitely sensitive to small changes in spatial relationships between tagged molecules.
- In practical applications, proteins of interest are genetically fused to fluorescent tags with appropriate spectral properties. Commonly used FRET pairs include cyan fluorescent protein (CFP) as the donor and yellow fluorescent protein (YFP) or mCherry as the acceptor. When the donor-tagged protein is excited, and the acceptor-tagged protein is in close proximity, energy is transferred, resulting in decreased donor fluorescence and increased acceptor fluorescence. This signal change can be detected and quantified using fluorescence microscopy, spectrometry, or fluorescence lifetime imaging microscopy (FLIM). In FLIM, the fluorescence lifetime of the donor is measured, which decreases in the presence of FRET, providing a direct and quantitative measure of molecular proximity independent of fluorophore concentration.
- FRET is extensively used to monitor dynamic processes in live cells, such as protein complex formation, receptor dimerization, conformational changes, and enzymatic activity. It allows researchers to visualize when and where interactions occur within the cellular context and can even detect transient or weak interactions that are difficult to capture by biochemical methods. Intramolecular FRET, in which donor and acceptor are fused to the same molecule, can also be used to study conformational changes or folding events.
- Despite its power, FRET requires careful experimental design. The fluorophores must be chosen to ensure significant spectral overlap between donor emission and acceptor absorption, and controls must be in place to correct for bleed-through and direct excitation of the acceptor. Furthermore, the orientation and relative position of the fluorophores can affect FRET efficiency, so linker lengths and tag positions may need optimization. Advanced imaging platforms such as confocal microscopy or FLIM systems are often used for high-resolution and quantitative analysis.
- In conclusion, FRET is a valuable and versatile technique that provides real-time, nanometer-scale insight into biomolecular interactions and structural changes in living cells. Its ability to monitor interactions in a native cellular environment has made it indispensable in the fields of cell biology, structural biology, and pharmacology.
