FLUORESCENCE RESONANCE
ENERGY TRANSFER
(FRET)
Anandhu Mohan
P170505
MSc Chemistry
25-10-2018
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FLUORESCENCE
FRAP
FRET
(Fluorescence
Recovery After
Photo bleaching)
(Fluorescence
Resonance
Energy Transfer)
FLIM
(Fluorescence
Lifetime imaging
Microscopy)
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Fluorescence Resonance Energy Transfer
(FRET)
■ Also Known As Förster Resonance Energy Transfer.
■ FRET is a distance dependant radiation less transfer of energy from an excited
donor fluorophore to a suitable acceptor fluorophore.
■ FRET is not the result of emission from the donor being absorbed by the acceptor.
■ In presence of suitable acceptor, the donor fluorophore can transfer its excited state
energy directly to the acceptor without emitting a photon.
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Mechanism
■ The energy transfer involves a donor fluorophore in an excited electronic state,
which may transfer its excitation energy to a nearby acceptor chromophore in a nonradiative fashion through long-range dipole-dipole interactions.
■ The theory supporting energy transfer is based on the concept of treating an excited
fluorophore as an oscillating dipole.
■ The dipole undergo an energy exchange with a second dipole having a similar
resonance frequency.
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Criteria for FRET
■ The fluorescence emission spectrum of the donor molecule must overlap the
absorption or excitation spectrum of the acceptor chromophore. The degree of
overlap is referred to as spectral overlap integral (J).
■ The two fluorophore (donor and acceptor) must be in the close proximity to one
another (typically 1 to 10 nm)
■ The transition dipole orientations of the donor and acceptor must be approximately
parallel to each other.
■ The fluorescence lifetime of the donor molecule must be of sufficient duration to
allow the FRET to occur.
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Efficiency of the FRET
■ Förster showed that the efficiency of the FRET process ( EFRET ) depends on the
inverse sixth power of the distance between the donor and acceptor pair (r).
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R
EFRET = 0ൗ(R6 + r6)
0
■ Where R0 is the Förster radius at which half of the excitation energy of donor is
transferred to the acceptor chromophore. Therefore Förster radius (R0 ) is referred to
as the distance at which the efficiency of energy transfer is 50%.
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Factors affecting FRET
■ Resonance energy transfer is not sensitive to the surrounding solvent shell of a
fluorophore.
■ The major solvent impact on fluorophores involved in resonance energy transfer is
the effect on spectral properties of the donor and acceptor.
■ Non-radiative energy transfer occurs over much longer distances than short-range
solvent effects, and the dielectric nature of constituents positioned between the
involved fluorophores has very little influence on the efficacy of resonance energy
transfer
■ FRET depends primarily on the distance between the donor and acceptor
fluorophore.
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Detection of
FRET
FLIM-FRET
Acceptor
photo
bleaching
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FLIM-FRET
■ The fluorescence lifetime of a fluorophore is affected by FRET, and in the presence
of a suitable acceptor, the lifetime of the donor will decrease.
■ This decrease in lifetime that FLIM-FRET seeks to measure.
■ FLIM-FRET is a robust method for calculating FRET efficiency, but it is costly both in
terms of financially (requiring a modulated light source and sensitive detectors) and
time.
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Acceptor photo bleaching
■ FRET can be measured by deliberately photo bleaching the acceptor molecule in
order that energy from the donor can no longer be transferred to the acceptor.
■ It leads to an increase in fluorescence of the donor molecule.
■ Then measure the fluorescence intensity of the donor molecule, bleach the
acceptor, and then re-measure the intensity of the donor.
■ The difference between these donor intensity measurements enables us to
calculate FRET.
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Applications of FRET
Applications of FRET
■ Structure studies
Structure
studies
Conformational
analysis
Interaction
between
molecules
Live cell
imaging
(FRETMicroscopy)
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Use of FRET to Study Association of
Proteins
■ FRET can be used to measure
protein association.
■ one monomer contains a
tryptophan residue, and the other a
dansyl group
■ Upon association FRET will occur,
which decreases the intensity of the
donor emission
■ The extent of donor quenching can
be used to calculate the donor-to
acceptor distance in the dimer
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FRET as a "spectroscopic ruler"
■ Measurement of the rate of FRET enables measurement of the distance between
the donor and the acceptor fluorophores.
■ Donor labelled at distinct sites of the molecule with different fluorophores, each of
these could donate energy to Acceptor.
■ The efficiency of FRET was measured by relative quantum yield in the absence and
presence of the acceptor.
■ Förster radius is determined by the spectral overlap of the donor emission with the
acceptor absorption.
■ Then the distance is calculated using the Förster equation.
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References
■ S. A. Hussain et. Al. An Introduction to Fluorescence Resonance Energy Transfer
(FRET) 2008
■ R Swift, S & Trinkle-Mulcahy, Laura. (2003). Basic principles of FRAP, FLIM and
FRET. Proc R Microsc Soc. 39.
■ Maria-Chantal Chirio-Lebrun, Michel Prats, Fluorescence resonance energy transfer
(FRET): theory and experiments ; Biochcfnical Education 26 (1998) 32(I-323
■ Ekaterina Sobakinskaya, Marcel Schmidt am Busch, and Thomas Renger, Theory of
FRET “Spectroscopic Ruler” for Short Distances: Application to Polyproline; J. Phys.
Chem. B 2018, 122, 54−67
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