Transcript FRET - cas.cz

CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Förster Energy Transfer
Excimer Fluorescence
Fluorescent Proteins
Martin Hof, Radek Macháň
Förster Resonance Energy Transfer - FRET
D*
A*
FRET
D
A
A fluorophore called donor (D) absorbs a photon and gets to its excited
state (D* - typically lowest vibrational level of S1 state)
If the energies of deexcitation processes of D* (such as fluorescence)
match excitation energies of another molecule (acceptor A) in its vicinity,
that means an overlap between emission spectrum of D and excitation
spectrum of A,
a radiationless energy transfer between D* and A can occur resulting in D
and A*. Acceptor can then emit fluorescence (if it is fluorescent).
Milestones in the theory of FRET
1918 J. Perrin proposed the mechanism of resonance energy transfer
1922 G. Cario and J. Franck demonstrate that excitation of a mixture of
mercury and thallium atomic vapors with 254 nm (the mercury resonance
line) also displayed thallium (sensitized) emission at 535 nm.
1928 H. Kallmann and F. London developed the quantum theory of
resonance energy transfer between various atoms in the gas phase. The
dipole-dipole interaction and the parameter R0 are used for the first time
1932 F. Perrin published a quantum mechanical
theory of energy transfer between molecules of the
same specie in solution. Qualitative discussion of the
effect of the spectral overlap between the emission
spectrum of the donor and the absorption spectrum
of the acceptor
1946-1949 T. Förster develop the first quantitative
theory of molecular resonance energy transfer
Basic properties of FRET
D*
A*
FRET
D
A
The interaction is of dipole nature and depends on the distance R of the
molecules and the orientation of their transition dipoles
The rate constant kET of FRET:
kET
1  R0 

 
D  R 
6
D is the lifetime of the donor in the absence of acceptor and R0 is a
constant for the donor-acceptor pair – Förster radius
Förster radius

R0[nm]  0.0211 n 4 QYD J k 2

16
n is the refractive index of the medium, QYD is the quantum yield of
donor, J is normalized overlap integral of donor and acceptor spectra and
k describes orientation of the dipoles
D
fD()
1,0
A()
A
Intensity
0,8
Tryptophan
DPH
0,6
0,4
0,2
0,0
250
300
350

400
450
500
550
[nm]
J   fD( )  A ( ) 4 d
0
600
Förster radius
k 2  cosfET  3 cosfD cosfA 
2
fET
the limits of k2 are from 0 to
4. If the molecules undergo
fast isotropic movement
D
fA
fD
R
A
(dynamic averaging) k2 = 2/3
Dynamic averaging (k2 = 2/3) is usually assumed in FRET analysis
and in tabulated values of R0.
Förster radius - examples
Some typical donor-acceptor pairs commonly used in structural
mapping of proteins, and their values of R0:
Donor
Acceptor
R0 [Å]
Fluorescein
Tetmethylrhodamine
55
IAEDANS
Fluorescein
46
Tryptophan
DPH
40
Fluorescein
Fluorescein
44
BODIPY
BODIPY
57
Energy transfer efficiency (E)
E 
kET
kET
  ki
i  ET
Where kET is the rate of energy transfer and ki of all other
deactivation processes.
Experimentally, E can be calculated from the fluorescence
lifetimes or intensities of the donor determined in absence and
presence of the acceptor.
 DA
E 1
D
or
IDA
E 1
ID
The distance dependence of the energy transfer efficiency (E)
16
1

R    1
E

R0
Where R is the distance separating the
centers of the donor and acceptor
fluorophores, R0 is the Förster radius.
The efficiency of transfer varies with the inverse sixth power of the
distance.
1
R0 in this example
was set to 40 Å.
When the E is 50%,
R = R0
Efficiency of transfer
0.75
0.5
Distances can generally
be measured between
~0.5 R0 and ~1.5R0
0.25
0
0
20
40
60
Distance in Angstrom
80
100
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
FRET
A
Determination of FRET efficiency
Intensity based:
•
Sensitized emission of the acceptor (provided it is fluorescent)
•
Decrease in intensity (quenching) of donor fluorescence
The main problem of intensity based approaches is the sensitivity
to donor and acceptor concentration
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 usually 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
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
D
 PB
E  1  DA
 PB
where PB is the intensity decay time due to photobleaching (I ~ exp(-t/PB))
Photobleaching measurement is not sensitive to concentration
and it does not require high temporal resolution – steady-state
instrumentation)
FRET and distance measurements
 FRET can be used to obtain structural maps of complex biological
structures, primarily proteins or other macromolecular assemblies.
 Measurements of energy transfer can provide intra- or
intermolecular distances for proteins and their ligands in the range
of 10-100 Å.
 FRET can detect change in distance (1-2 Å) between loci in
proteins, hence it is a sensitive measure of conformational
changes.
 The donor and acceptor must be within 0.5 R0 - 1.5 R0 from each
other.
 These measurements give the average distance between the two
fluorophores. When measuring a change in distance, the result
gives no indication of which fluorophore moves.
 Experiments can be done with different donor-acceptor pairs. If the
same distance is obtained, the result is very likely correct.
 Quantitative analysis is restricted to the cases where only a few
donors and acceptors are present
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).
Fluorophores for FRET in proteins
Synthetic organic dyes (BODIPY, Dansyl, AEDANS, …) attached to the
protein for example via amine groups (N-terminus, lysine) or via
sulfhydryl groups (cysteine)
Aromatic amino acid residues (Trp, Tyr, Phe).
Possible FRET pairs are for example:
Tyr – Trp or Phe - Tyr
Fluorescent proteins (GFP, mCherry, …) are expressed in some organisms
and can be genetically encoded to be expressed at desired locations of
other proteins of other organisms. Mutations allow expressions of
proteins of various spectral characteristics of fluorescence.
 FRET pairs
Green Fluorescent Protein (GFP)
• Appears in sea organisms, Structure of
GFP of jelly fish Aequorea victoria is
know (1996)
• 13 ß-sheets forming the ß-barrel
• an a-helix inside the ß-barrel and a
heterocyclic chromophor
in vivo excitation of GFP
• Attack increases cellular
Ca2+-concentration
• Calcium binds to Aequorin
• CO2 is released
• Energy of excited Aequorin
is transferred to GFP
• GFP fluoresces
Formation of the Chromophore
• formed by AS 65-67:
Ser-Tyr-Gly
• p-Hydroxybenzyliden-imidazolidon
• Isolated chromophore
is not fluorescent
folding
G
Y
cyclization
-H20
S
Fluorescence and Absorption (Wild-Type)
• Abs. at 395 and 475 nm
• Fluorescence at 509 nm
1,0
• Fluorescence almost
0,8
Intensity
independent on ex
0,6
0,4
0,2
0,0
340 360 380 400 420 440 460 480 500 520 540 560 580 600
[nm]
Förster-Cycle
• A type of excited state
reaction
• Phenols are not acid in
the S1-state
• fs-spectroscopy: after
395 nm excitation, redshift of fluorescence to
509 nm during 10 ps
Mutations lead to 6 classes of FP
• Class 1:
Wild–type
• Class 2: Phenol-anion
EGFP
• Class 3: neutral
Phenol in S0
t203i
Mutations lead to 6 classes of FP
• Class 4 : - interaction
 yellow emission
YFP
• Class 5 : Trp replaces
Phenol  cyan em.
CFP
• Class 3: Imidazole
replaces Trp  blue em.
BFP
Cameleon proteins – FP based biosensors
A biosensor for intra-cellular
free Ca2+. CFP and YFP are
coupled through a Ca2+binding protein calmodulin,
which undergoes a dramatic
structural change upon Ca2+
binding. The juxtaposition
of FP results in FRET – CFP
is donor and YFP acceptor.
-Ca
2 +
donor excitation at 440 nm
+ Ca
2 +
480
535
CFP
YFP
Fluorescence emission in nm
Truong et al. Nat Struct Biol, 8: 1069
E1
Excimer fluorescence
Excimer stands for excited dimer. The excimer has a different spectrum
than the monomer – red shifted. A typical excimer forming fluorophore is
pyrene (Förster 1954)
M* + M → (MM)*
1.
2.
3.
4.
2 mM (Argon)
2 mM (air)
0.5 mM (Argon)
2 M (Argon – to
remove oxygen)
Pyrene in ethanol
Note: If an excited complex of two different molecules is formed, it should be called
exciplex, however even then the term excimer is often used (some excimer lasers)
Pyrene excimer fluorescence
Application: An assay for membrane fusion. Fusion results
in dilution of pyrene in the membrane → decrease in the
ration of excimer/monomer fluorescence intensity
E2
Pyrene excimer and lipid diffusion in bilayers
Pyrene excimer formation can be used to determine diffusion coefficient
(D) of lipids in bilayers of vesicles (using lipids labelled with pyrene)
kE
M* + M → (MM)*
kE = 2 D
cM – monomer surface
concentration
Excimer formation competes with fluorescence of monomer and nonradiative deexcitation of monomer
monomer fluorescence:
IM  n *
kf
 n * kf  M
kf  knr  kE cM
E
k
kE cM
E
f
excimer fluorescence: I  n

n
*
k
E
E
f E
E
E
kf  knr
kf  knr  kE cM
IE
kfE
 k E cM  E
IM
kf
constant for a fluorophore, ≈ 10 for
pyrene
Determination of D via kE
Acknowledgement
The course was inspired by courses of:
Prof. David M. Jameson, Ph.D.
Prof. RNDr. Jaromír Plášek, Csc.
Prof. William Reusch
Financial support from the grant:
FRVŠ 33/119970