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Fluorescence methods in life sciences
Ctirad Hofr
Fluorescence resonance
energy transfer
Energy transfer
Overview of the lecture
1. The origin of resonance energy transfer
2. Characteristic values of FRET
3. FRET applications for biomolecules
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1. Excitation energy transfer
• Electron transfer of energy takes place by radiative or nonradiative
mechanisms.
• The radiative (trivial) energy transfer occurs when the excited
donor molecule emits radiation which is subsequently reabsorbed
by the acceptor molecule.
The photon is exchanged.
• Excitation by non-radiative energy transfer (fluorescence
resonance energy transfer, FRET) occurs when only donor
molecules absorb in the mixture of molecules. As a result, excited
acceptor molecules that don't absorb the excitation radiation.
During this transfer of energy the donor does not emit the light .
There is no photon exchanged!
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What is resonance energy transfer
good for?
• Conformation changes
• Binding of a ligand and
a receptor
• Cleavage of a protein/DNA
• Membrane fusion
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Resonance energy transfer
• The analogy with the energy transfer between
tuners
• In the case of molecules, transfer of energy
occurs due to dipole-dipole interactions
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Transition dipole moment
• Transition dipole moment is a quantum mechanical issue. It is not a real
dipole moment. It is given by immediate state of electron shell of the molecule.
The size of the dipole moment of transfer indicates the ability of the given
molecule state to absorb or emit the light. The direction of the dipole moment of
transfer indicates the direction in which the light is preferably absorbed or
emitted by the molecule.
• The molecules preferentially absorb the light whose electric component
oscillates in the same plane as absorption transition dipole of electron into a
higher energetic level.
• Molecules preferably emit light in the same plane as the emission transition
dipole of electron transition into a lower energetic level.
Absorption
Emission
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Interaction of transfer dipole
moments of Donor and Acceptor
Donor Acceptor
Absorption of donor
Emission of donor
Absorption of acceptoru
Emission of acceptoru
Abs
λ(nm)
Fluorescence
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FRET – non-radiative energy
transfer
• In the presence of a
suitable acceptor, a donor
can transfer energy of the
excited state directly to
the acceptor without
emission of a photon.
• The excited acceptor then
emits the energy that was
originally absorbed by the
donor.
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Fluorescence resonance energy
transfer (FRET)
Resonance energy transfer can be characterized by the
rate constant (kDA), which expresses the probability of
transfer; the dipole-dipole energy transfer is the
determining component for that Förster formula has been
derived (in the case of the weak bond when mutual donor
and acceptor interactions do not affect the optical spectra)
in the form
τD – time decay of donor fluorescence , R0 – the distance at which the
probability of energy transfer is equal to the probability of internal deactivation of
the excited state of a molecule, r – the distance between the donor and the
acceptor.
Resonance energy transfer is highly dependent on the distance of
the donor and acceptor.
6
01








⋅=
r
R
k
D
DA
τ
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Transfer efficiency E
Is determined by the relative amount of photons
that are absorbed by the donor and
subsequently emitted by the acceptor.
D
DA
F
F
E −=1
It is measured by determining of the relative intensity
of donor fluorescence in the absence (FD) and in
the presence of acceptor (FDA).
Rapidly decreases with the 6 power of the distance!
)(
)(
1
rk
rk
E
DAD
DA
+
= −
τ 66
0
6
0
rR
R
E
+
=
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R0 Förster distance
• R0 is the distance of the donor and acceptor at which the transfer
efficiency E is 50% = half
• Size of R0 is usually 2 – 6 nm, which is comparable with the size of
biomolecules
The dependence of transfer efficiency E on distance r is the highest (the fastest
change) in the case that the donor and acceptor distance is close to R0.
R0 indicates middle distance on which donor-acceptor pair can communicate –
R0 is an unique characteristic of the pair of fluorophores
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The dependence of transfer efficiency on
distance
r = 0.1 x R0
r = 0.5 x R0
r = 2.0 x R0
r = 4.0 x R0
66
0
6
0
rR
R
E
+
=
E = 0.999999
E = 0.984615
E = 0.015384
E = 0.000244
Suitable distances for measurement: 0.5 - 2 R0
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What does R0 depend on?
• On the orientation of the transfer dipole moments for the donor emission
and acceptor absorption (the average value of 2/3 is used for factor κ2)
• On the optical properties of environment (refractive index of water nH20=
1.33)
• On the quantum yield of the donor QD
• On the degree of overlap J (λ) of the donor emission spectrum and the
acceptor absorption spectrum
( )
o6/142
0 A)(v)(211.0 λκ JQnR D
−
=
Abs
λ(nm)
Fluorescence
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Indicative factor κ2 dependence on the
orientation of transfer dipole moments
For the random arrangement of
transfer dipole moments
κ2 = 2/3
This value is commonly used in
common practice.
In the case that the transfer dipole
moments are perpendicular κ2= 0
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Dependence of R0 on spectral overlap
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Distance measurement using
FRET
• Melittin is the most important component of honeybee venom
(damaging the cell structure, decomposes the white and red blood
cells, it causes death of cells). Its anti-inflammatory effects are used
in the treatment.
• Melittin consists of 26 AK, tryptophan at position 19 was the donor,
• Dansyl at the N-terminus was the acceptor
• After separate spectral characterization of melittin with Trp as the
donor and Dansyl as the acceptor the value R0=2.36 nm was
determined .
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Determination of the Trp distance from
the N-terminus of melittin using FRET
• Based on the decrease in fluorescence intensity in the
presence of the acceptor it is possible to determine the
transfer efficiency and hence donor (Trp) distance from the
acceptor (N-terminal).
• r = 2.44 nm
D
DA
F
F
E −=1
66
0
6
0
rR
R
E
+
=
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FRET advantages
• Efficiency of energy transfer is not dependent
on environment between the donor and acceptor
• Measurement of intensity ratio enables the use of
FRET analysis independently on the
concentration
• For most applications, it is not necessary to
know R0
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FRET allows to distinguish between
multiple variants of protein folding
• apoA-I protein regulates cholesterol metabolism.
• apo A-I binds to a lipid membrane
• two possible arrangements of proteins in the membrane were
designed
• structural methods can not be used because of the flexibility of
lipids
• one of the chains of apoA-I proteins was labeled by fluorescein
(Donor) and second one by tetramethylrhodamine (Acceptor)
• Firstly, the emission spectrum of the donor itself in
one chain of ApoA-I was measured
• Decrease of the intensity of donor fluorescence was
observed after addition of the second chain with the
acceptor
• There was a significant resonance energy transfer
• This proved a relatively small distance between the
donor and the acceptor
• It was confirmed belt conformation of ApoA-I
http://pubs.acs.org/doi/abs/10.1021/la402727a
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FRET between CFP and YFP
CFP YFP
http://www.cytographica.com/animations/index.html
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FRET quenching by photobleaching
CFP YFP
http://www.cytographica.com/animations/index.html
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Protein folding in vivo
• ApoE4 is associated with Alzheimer's
disease and binds to nerve cells
• ApoE3 has a similar sequence of AK,
but does not bind to nerve cells
• It has been suggested that the binding
ability to the nerve cells is related to the
different arrangement of domains of the
ApoE proteins
• For verifying nerve cells were
transfected with constructs of ApoE
• FRET analysis showed that ApoE4 in
conformation with associated domains
binds to nerve cells, while ApoE3 does
not have its domains in close proximity
and does not bind to the nerve cells
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FRET sensors
• Estrogen detection
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Monitoring of protein
phosphorylation
Monitoring of insulin processing
Fluorescence
Insulin receptor is a tyrosine kinase
which phosphorylates tyrosine in the
recognized sequence, which is
subsequently bound to the SH2 domain
of another protein.
This binding causes the CFP and YFP
approach and induces FRET.
The protein construct was expressed in
cells.
Transfer energy was increased
gradually after exposure of cells to 100
nM insulin.
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Monitoring of morphological
changes of cells
The most of GTP is processed in the green region
of FRET and the cell also moves in this direction.
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Ribozyme cleavage
• Parts A and B get near upon
binding of the ribozyme to a
substrate resulting in increased
FRET
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Detection of the presence of c-fos
mRNA
• c-fos mRNA is a
transcription factor that
affects cell cycle and
differentiation
• FRET probes were used to
detect the presence of c-fos
mRNA in the cell culture
• The presence of c-fos
mRNA in activated cells
(left) was demonstrated
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Membrane fusion
• Individual membranes
contain a donor or an
acceptor
• in the case of membrane
fusion we observe FRET
Application of FRET microscopy
FRET was applied for visualization of lipid bilayer vesicle fusion:
Intermediates Captured by High-Speed Microfluorescence Spectroscopy
Lei, G and MacDonald, R.C., Biophys J. 2003
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Literature
• Lakowicz J.R.: Principles of Fluorescence
Spectroscopy. Third Edition, Springer +
Business Media, New York, 2006.
• Fišar Z.: FLUORESCENČNÍ SPEKTROSKOPIE
V NEUROVĚDÁCH
http://www1.lf1.cuni.cz/~zfisar/fluorescence/Default.htm
Graphics from the book Principles of Fluorescence for the purpose
of this lecture was kindly provided by Professor J.R.Lakowitz.
FRET animations at full resolution were kindly provided by
Dr. Joseph T. E. Roland
Acknowledgment
Intrinsic fluorescence of proteins
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