Protein-protein interactions are critical for cellular function and a major target class for drug discovery, yet they are difficult to investigate. FRET (Förster or Fluorescence Resonance Energy Transfer) is the non-radiative transfer of energy from an excited fluorophore (donor) to another fluorophore (acceptor). FRET only occurs when the molecules concerned are in very close proximity. This makes it a valuable technique for studying interactions between molecules, such as proteins, in solution or in cells. Exciting the donor and then monitoring the relative donor and acceptor emissions, either sequentially or simultaneously, makes it possible to determine when FRET has occurred.
Detection of FRET can be used to quantify when and where two or more biomolecules interact. As the energy transfer occurs only over distances between 1-10 nm, the distance between interacting proteins can be measured with a precision beyond the optical resolution of the light microscope (~0.25 µm). As a result, FRET is a useful tool to quantify molecular dynamics, such as protein-protein interactions, protein-DNA interactions, and conformational changes of molecules in living cells.
A common combination of fluorophores for studying protein-protein interactions in cells of interest is cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor-acceptor pair.
When CFP is excited and the two molecules are within 10 nm of each other, energy may be transferred from the excited CFP to the YFP causing the YFP to emit yellow light, which is then detected independently of the blue light that excited CFP
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Fluorescence Resonance Energy Transfer (FRET)