1. Förster Resonance Energy Transfer (FRET)
Marek Scholz

2. Activation of prior knowledge
What is rate constant, what is its unit and how deactivation rate relates to the excited state lifetime?
What is Stokes shift?
What wavelengths (approximately) have blue, green and red color?
How the quantum yield (QY) of fluorescence is defined?
What is typical for molecular structure of fluorophores?
What is CFP and YFP?
What is usually denoted as S1 state? Why S?
By which processes the S1 state can be depleted?
Which processes can cause fluorescence depolarization?

3. What is FRET? D A D D A Forster Resonance Energy Transfer
Two dyes, donor and acceptor (D, A)
Donor absorbs photon. If the acceptor is close enough (<10nm), then donor can transfer the excitation energy to the acceptor.
It is an electric dipole-dipole near-field interaction, it is NOT due to photon emission and reabsorption!
FRET D A D
FRET D A
5 nm

4. What is FRET good for? proximity indicator spectroscopic ruler
protein-ligand interactions, complex formation, bioaffinity assays, DNA hybridization indicator
conformational changes
spectroscopic ruler
distances within the biomolecules can be determined based on the efficiency of FRET
Biomolecules have to be labelled with fluorophores at strategic sites

5. What is FRET good for?

6. Under what conditions FRET occurs?
The interaction is of dipole nature (coupled oscillators in resonance)
It depends on the distance R of the molecules and the orientation of their transition dipoles. There also has to be spectral overlap of donor emission and acceptor absorption.

7. distance dependence of FRET
kET
at certain distance
kET = kD = 1/tauD
knr + kr = kD = 1/tauD
The rate constant kET of FRET:
tD is the lifetime of the donor in the absence of acceptor and R0 is a constant for the donor-acceptor pair – Förster radius. It corresponds to the distance where FRET is 50% efficient.

8. FRET efficiency
Where kET is the rate of energy transfer and ki of all other deactivation processes.
Distances can generally be measured between ~0.5 R0 and ~1.5R0

9. Förster radius R0 Förster radius is specific for a donor-acceptor pair
n is the refractive index of the medium, ΦD is the quantum yield of donor, J is normalized overlap integral of donor’s emission and acceptor’s absorption spectra, and k describes orientation of the dipoles.
eA(l) D
fD(l) A
DPH
Tryptophan

10. orientation factor
If the molecules undergo fast isotropic movement (dynamic averaging) k2 = 2/3

11. Forster radius examples

12. Homo energy transfer
Energy transfer between molecules of the same fluorophore
fluorescein
There exists en overlap between the excitation and emission spectrum of a fluorophore
Homo energy transfer is responsible for:
self-quenching of fluorophores at high concentration
decrease in anisotropy of fluorescence at high fluorophore concentrations (Gaviola and Prigsham 1924) D A
FRET

13. Determination of FRET efficiency Determination of FRET efficiency
Intensity based:
Decrease in intensity (quenching) of donor fluorescence
Sensitized emission of the acceptor (provided it is fluorescent)
Donor fluorescence in the presence and absence of acceptor
NBD-PC liposomes  TexasRed-temporin B (membrane active peptide)

14. Determination of FRET efficiency
Kinetic based:
Decrease in lifetime (quenching) of donor fluorescence
Fluorescence decay of acceptor - It contains a rise in the initial phase corresponding to the kinetics of donor deexcitation by FRET (a component with “negative amplitude” in the fitted decay)
Kinetics of donor photobleaching
The use of donor fluorescence is preferred, because the acceptor is usually to some extent excitable by the excitation wavelength of the donor – only a part of acceptor fluorescence is a result of FRET

15. Photobleaching of donor
Photobleaching is a decrease in fluorescence intensity due to permanent inactivation of the fluorophores.
It is usually caused by excited state reactions of the fluorophore in triplet state (for example with oxygen). Photobleaching is observed mainly at high excitation intensities when a significant fraction of molecules undergoes intersystem crossing.
FRET represents an additional deexcitation channel and, thus decreases the probability of intersystem crossing and photobleaching. The decrease of intensity due to photobleaching is, therefore, slower
Fluorescence intensity
Time (sec)
where tPB is the intensity decay time due to photobleaching (I ~ exp(-t/tPB))
Photobleaching measurement is not sensitive to concentration and it does not require high temporal resolution – steady-state instrumentation)

16. FRET concepts in protein science
FRET between a donor and acceptor, each attached to a different protein, reports protein–protein interaction.
Two fluorophores are attached to the same protein, where changes in distance between them reflect alterations in protein conformation, which in turn indicates ligand binding. Abrogation of intramolecular FRET can be used to indicate cleavage.
A protein or antibody fragment (blue) binds only to the activated state of the protein. The antibody fragment bears a dye which undergoes FRET when it is brought in close proximity to the dye on the protein. In some examples, the domain is part of the same polypeptide chain as the protein (dashed line).

17. further FRET concepts

18. single molecule FRET