1. Förster Energy Transfer
CZECH TECHNICAL UNIVERSITY IN PRAGUE
FACULTY OF BIOMEDICAL ENGINEERING
Förster Energy Transfer
Excimer Fluorescence
Fluorescent Proteins
Martin Hof, Radek Macháň

2. Förster Resonance Energy Transfer - FRETD* 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).

3. 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
T. Förster develop the first quantitative theory of molecular resonance energy transfer

4. Basic properties of FRETD* 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:
tD is the lifetime of the donor in the absence of acceptor and R0 is a constant for the donor-acceptor pair – Förster radius

5. Förster radius
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
fD(l)
eA(l) D A
DPH
Tryptophan

6. Förster radius
fET D fA
the limits of k2 are from 0 to 4. If the molecules undergo fast isotropic movement (dynamic averaging) k2 = 2/3 fD A R
Dynamic averaging (k2 = 2/3) is usually assumed in FRET analysis and in tabulated values of R0.

7. 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 46
Tryptophan
DPH 40 44
BODIPY 57

8. Energy transfer efficiency (E)
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. or

9. The distance dependence of the energy transfer efficiency (E)
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.
0.25
0.5
0.75 1 20 40 60 80
100
Distance in Angstrom
Efficiency of transfer
R0 in this example was set to 40 Å.
When the E is 50%, R = R0
Distances can generally be measured between ~0.5 R0 and ~1.5R0

10. 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

11. 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

12. 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
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)

13. 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 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 R 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

14. 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).

15. 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

16. 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

17. 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

18. Formation of the Chromophore
formed by AS 65-67: Ser-Tyr-Gly
p-Hydroxyben-zyliden-imidazolidon
Isolated chromophore is not fluorescent
folding
cyclization G Y
-H20 S

19. Fluorescence and Absorption (Wild-Type)
Abs. at 395 and 475 nm
Fluorescence at 509 nm
Fluorescence almost independent on lex

20. Förster-Cycle A type of excited state reaction
Phenols are not acid in the S1-state
fs-spectroscopy: after 395 nm excitation, red-shift of fluorescence to 509 nm during 10 ps

21. Mutations lead to 6 classes of FP
Class 1: Wild–type
Class 2: Phenol-anion
Class 3: neutral Phenol in S0
EGFP
t203i

22. Mutations lead to 6 classes of FP
Class 4 : - interaction  yellow emission
Class 5 : Trp replaces Phenol  cyan em.
Class 3: Imidazole replaces Trp  blue em.
YFP
CFP
BFP

23. 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.
donor excitation at 440 nm
Fluorescence emission in nm
CFP
YFP
Truong et al. Nat Struct Biol, 8: 1069

24. Excimer fluorescence M* + M → (MM)*
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)*
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)

25. 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

26. 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
cM – monomer surface concentration
M* + M → (MM)*
kE = 2p D
Excimer formation competes with fluorescence of monomer and non-radiative deexcitation of monomer
monomer fluorescence:
excimer fluorescence:
constant for a fluorophore, ≈ 10 for pyrene
Determination of D via kE

27. 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