"Resonance energy transfer is the radiationless transmission of an energy quantum from its site of absorption to the site of its utilization in the molecule, or system of molecules, by resonance interaction between chromophores, over distances considerably greater than interatomic, without conversion to thermal energy, and without the donor and acceptor coming into kinetic collision. The donor is the dye that initially absorbs the energy, and the acceptor is the chromophore to which the energy is subsequently transferred". (from: Van Der Meer, B.W., Coker, G.III, and Chen, S.-Y. (1994) Resonance Energy Transfer Theory and Data, VCH, New York).
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To measure distances or changes in distance, you need to specifically and uniquely label your molecule of interest with a donor and an acceptor probe. However, multiple labeling can still give you some informations about conformational changes occuring in your molecule.
The donor probe is a fluorescent molecule. When light excites the fluorophore
at an appropriate wavelength (250-500 nm) its electrons jump from the ground
to a higher vibrational level (S,
etc). Within picoseconds these electrons decay to the lowest of these vibrational
and then decay more slowly (nsec) to one of the S
states and a photon of light is emitted whose wavelength is longer than
the exciting wavelength.
Figure 1 is modified from dos Remedios and Moens, 1995.
For FRET to occur clearly:
The acceptor probe can be fluorescent or non-fluorescent.
Obviously, you need an instrument to measure either the fluorescence intensity or the lifetime of your fluorophore!!! ....and a spectrophotometer to determine your molecule and label concentrations as well as to record the absorption spectrum of the acceptor probe.
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Once the conditions to observe FRET are met, and the donor probe is excited at the appropriate wavelength, decay as donor fluorescence and energy transfer to the acceptor will compete for the decay of the excitation energy and can be described by the following scheme:
where D refers to the donor probe and A to the acceptor probe (see figure 1), Kd and Ka are the radiative decays of the donor in the absence of the acceptor and of the acceptor, respectively. Kt is the rate of energy transfer(radiationless), hve, hvd and hva are the photon energies of the donor excitation, of the donor fluorescence and of the acceptor fluorescence. Kdi and Kai are radiationless decay constants. (from Van der Meer et al., 1994)
The quantum yield (Q) of the donor is defined as the ratio of the number of photons emitted to the number absorbed, a parameter which depends on the immediate environment of the probe. The donor quantum yield in the presence of transfer (Qda) and in the absence of transfer (Qd) are
FRET efficiency (E) can be obtained by measuring the fluorescence intencities of the donor with acceptor (Qda) and without acceptor (Qd).
FRET efficiency can also be measured using the lifetime of the donor in presence (Tda) and absence of the acceptor probe (Td)
The relationship between the transfer efficiency and the distance between the two probe (R) illustrated in figure 2 is given by the equation:
Figure 2. The solid curve represents the relationship between the efficiency of the fluorescence resonance energy transfer and the distance separating the donor and the acceptor. The importance of this relationship is that there is a limited range of donor-acceptor distances which can be probed by any particular donor-acceptor pair.
Ro can be calculated using
where Qd is the quantum yield of the donor, n is the refractive index of the medium and is generally assumed to be 1.4 (range 1.33-1.6) for proteins, Nav is Avogadro's number (Nav= 6.02 x 10 per mole), K is the orientation factor and J is the overlap integral (from Van der Meer et al., 1994).
The overlap integral J represents the degree of overlap between the donor fluorescence spectrum and the acceptor absorption spectrum and is given by
Where is the wavelength of the light, () is the molar extinction coefficient of the acceptor at that wavelength, and f() is the fluorescence spectrum of the donor normalised on the wavelength scale.
where F() is the donor fluorescence per unit wavelength interval (from Van der Meer et al., 1994).
The orientation factor is defined as
where is the angle between the donor emission transition moment and the acceptor absorption transition moment, and and are the angles between the donor-acceptor connection line and the donor emmission and the acceptor absorption transition moments, respectively. Kappa square varies between 0 and 4. In the Förster equation kappa square assumes a numerical value of 2/3 provided that both probes can undergo unrestricted isotropic motion. For a discussion see dos Remedios and Moens, 1995.
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