Förster Resonance Energy Transfer (FRET)

A commonly used spectroscopic technique to estimate very small distances within or between macromolecules. Sometimes incorrectly referred to as Fluorescence Resonance Energy Transfer, FRET is the radiationless tranfer of energy from one molecule to another.

The experiment is often set up with a donor and an acceptor fluorescent probe. The donor and acceptor are selected such that the absorption spectrum of the acceptor is in the same wavelength region as the emission spectrum of the donor. When the donor and acceptor are in close enough proximity, energy is transferred through space between them. Using a wavelength that excites only the donor, energy transfer is observed by fluorescence emitted by the acceptor.

note: It is a common misconception that energy gets transferred between donor and acceptor by a photon. FRET is NOT a photon emitted by the donor that then collides with the acceptor. It is radiationless energy transfer. A good analogy is two tuning forks. If the vibration of one fork is of a period that it can resonate with another, energy will slowly dissipate from one, while the other begins to vibrate.

Factors that affect transfer efficiency

The efficiency of transfer between donor and acceptor is a function of many parameters:
  • quantum yield of both the donor and acceptor. Intuitive, because those fluorophores with a large number of competing processes depopulating the excited state are less likely to donate or accept energy.
  • orientation. sometimes called kappa - the critical component of energy transfer is that the oscillating dipole of the excited donor and the transition dipole of the acceptor ground state are aligned. If donor and acceptor are rigidly held in good alignment, then the efficiency is much higher. For cases where the rotation of donor and acceptor are considered to be random, a kappa2 value of 2/3 is usually used.
  • refractive index. physical property of the environment which affects the absorbance and emission properties of the fluorophore.
  • R0. distance constant that depends on the degree of overlap of the donor emission and the acceptor absorption spectrum. At R0 angstroms, the energy transfer efficiency is 50% of maximum.

How is it measured?

There are two ways to detect FRET. One is by a lowering of the intensity of the donor, and the other is the observed emission of the acceptor. If you have a certain amount of donor, with its natural intensity, adding acceptor will reduce the quantum yield as lost by FRET. However, this is a problematic way of observing FRET. Fluorescence is sensitive to a large number of characteristics of the environment including polarity, refractive index, oxygen concentration, etc. If any of these are affected by the addition of the acceptor, then one might be fooled into thinking energy transfer is occuring when it is not. However, emission from the acceptor is the best evidence of FRET. Since you are not exciting the acceptor directly, the only source of energy for it is the donor. Thus an emission spectrum of a donor vs a donor+acceptor should show a reduction in the intensity of the donor, and an appearance of a new spectrum in the range of the acceptor.

What are the applications?

The distance scales measured by FRET are on the order of angstroms (10-10m). This is a good scale for looking at distances in proteins. Chemically attaching a donor and acceptor to two different parts of a protein can then tell you how far apart they are. This can give you a rough idea of the dimension and shape of the protein, particularly useful when the protein is not amenable to direct structural characterization by X-ray crystallography or Nuclear Magnetic Resonance (NMR). It can also be used to detect interactions between proteins on a microscope. If you are interested in dimerization of two components on the surface of a cell, label one component with the donor, the other with the acceptor, and then look for acceptor emission through FRET. This gives you an additional piece of information over standard fluorescence labelling. Although the resolution of a microscope is not on the angstrom scale, acceptor fluorescence is good evidence of intermolecular contact in this distance range. FRET has been used to study structural states of hemoglobin, because the iron binding heme is a great FRET acceptor. Green Fluorescent Protein (GFP) has also been shown to exhibit FRET. Mutants of different colors can be used as donors or acceptors inside of live cells. Tryptophan has long been used as a fluorescence donor to chemically attached acceptors such as dansyl, allowing one to monitor dynamic motion of proteins, or folding/unfolding events. FRET may start to play a role in the development of nanomachines, where the detection of angstrom scale association events can help one visualize whether nano-assembly is successful.

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