FRET(Fluorescent Resonance
Energy Transfer)
The problem
• The use of fluorescent probes (ex. GFP)
permits direct observation of the dynamic
properties of specific proteins in live cells .
• But, It is difficult to observe functional
information like protein-protein interaction
using fluorescent probes.
• when proteins are labeled with different
fluorophores, the optical resolution of light
microscopes limits the detection of protein
proximities to about 0.2 µm.
What is FRET?
• FRET is a process that shifts energy from
an electronically excited molecule(donor)
to a neighboring molecule(acceptor).
• If the two fluorophores are close enogh,
then excitation of the first molecule
(Donor) results in fluorescence emission
of the second molecule (Acceptor).
What is FRET?
CFP is in close to YFP.
(1-10 nm)
No FRET Signal
•CFP is excited by light and emits light
•CFP is more than 10 nm distant from YFP
•YFP is not excited and does not emit light
FRET Signal
•CFP is excited by light but does emit little light
•CFP is in close proximity (1-10 nm) to YFP
•YFP is not excited by light but does emit light
• transfer its excitation energy to a nearby acceptor
chromophore in a non-radiative fashion through longrange dipole-dipole interactions.
Fluorophore pair for FRET
• The donor emission spectrum must overlap significantly
with the acceptor excitation spectrum.
Fluorophore pair for FRET
• The excitation light for the donor must
not directly excite the acceptor.
Donor
Excitation
Acceptor
Excitation
CFP
440nm
480nm
YFP
520nm
535nm
BFP
365nm
460nm
GFP
488nm
535nm
CFP
440nm
480nm
dsRed1
560nm
610nm
FITC
488nm
535nm
Cy3
525nm
595nm
Cy3
525nm
595nm
Cy5
633nm
695nm
GFP
488nm
535nm
Rhodamine 543nm
595nm
Donor
Emission
Donor
Acceptor
Emission
Acceptor
• A donor can directly transfer its excitation energy to
an acceptor through long-range dipole-dipole
intermolecular coupling.
– A theory proposed by Theodor Förster in the late
1940s
– Resonance energy transfer is a non-radiative
quantum mechanical process (no collision, heat)
When energy transfer occurs, the acceptor molecule
quenches the donor molecule fluorescence, and if
the acceptor is itself a fluorochrome, increased or
sensitized fluorescence emission is observed
Rate constant , K
• Rate constant for energy transfer(Kt)
•
𝑄𝐷𝐽
𝐾𝑡 ~
𝜏𝐷
×
1
𝑅6
× 𝜅2
– 𝐾𝑡 transfer rate
– 𝑄𝐷 quantum yield of donor
– 𝐽 spectral overlap of donor emission and
acceptor absorption
– 𝜏𝐷 fluorescence lifetime of donor
– R distance between donor and acceptor
– 𝜅2 orientational factor
FRET Efficiency
• Efficiency of Energy Transfer
𝑑𝑒𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑏𝑦 𝑒𝑛𝑒𝑟𝑔𝑦 𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟
=
𝑑𝑒𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑏𝑦 𝑎𝑙𝑙 𝑜𝑓 𝑡ℎ𝑒 𝑝𝑎𝑡ℎ𝑤𝑎𝑦𝑠
E = kT / (kT + kr + knr)
kT = rate of transfer of excitation energy
kr = rate of fluorescence(radiation)
knr = sum of the rates of all other deexcitation
processes (nonradiation)
R0 determination
• E = kT / (kT + kr + knr)
• Let,
•
R0 : distance between the donor and the acceptor
at which 50% of FRET efficiency takes place.
Then, E = 1 1+ 𝑅 6
𝑅0
R0 (Forster distance) = 9.78 x 103(n-4*fd*k2*J)1/6 Å
depends on J(spectral overlap)
Structural design
of various FRETbased biosensors.
a.
The interaction of two proteins
can be dynamically detected by
FRET
b.
Molecular cleavage by protease
will be translated into loss of FRET.
c.
An intramolecular probe consists
of sandwiching two domains
between CFP and YFP, which can
interact after phosphorylation or
binding to calcium, resulting in a
change in FRET.
d.
An intramolecular probe consists
of CFP, YFP and a protein/domain,
which permits conformational
change by binding to another
biomolecule, leading to a change
in FRET
FRET quantification
• FRET quantification is mostly based on
measuring changes in fluorescence
intensity or fluorescence lifetime upon
changing the experimental conditions.
Ex>a microscope image of donor emission
with normal acceptor and with acceptor
bleached.
Various FRETs
• CFP-YFP pairs
-most popular FRET pair for biological use.
-CFP(cyan fluorescent protein)
-YFP(yellow fluorescent protein)
both are GFP variants.
They can be easily attached to a host protein
by genetic engineering
Various FRETs
• BRET(Bioluminescence Resonance Energy
Transfer)
-FRET require external illumination to initiate the
fluorescence transfer.
-BRET use bioluminescent luciferase rather than
CFP(donor) to produce an initial emission compatible
with YFP.
Various FRETs
• Homo FRET
-to examine the interactions between two, or more
proteins of the same type.
-both the acceptor and donor protein emit light
with the same wavelenths.
-by FRET anisotropy imaging, measuring FRET is
available.