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

Förster Energy TransferExcimer FluorescenceFluorescent Proteins

Martin Hof, Radek Macháň

CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

Page 2: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Förster Resonance Energy Transfer - FRET

D

D*

A

A*

FRET

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).

Page 3: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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

Page 4: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Basic properties of FRET

D

D*

A

A*

FRET

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: 6

0

D

1

RR

kET

D is the lifetime of the donor in the absence of acceptor and R0 is a constant for the donor-acceptor pair – Förster radius

Page 5: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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 describes orientation of the dipoles

612D

40 0211.0]nm[ JQYnR

0

4AD d)()( fJ

250 300 350 400 450 500 550 6000,0

0,2

0,4

0,6

0,8

1,0

Inte

nsity

[nm]

AfD() A()D

Tryptophan

DPH

Page 6: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Förster radius

the limits of 2 are from 0 to 4.

If the molecules undergo fast

isotropic movement (dynamic

averaging) 2 = 2/3

D

A

DA

ET

R

2AD2 coscos3cos ET

Dynamic averaging (2 = 2/3) is usually assumed in FRET analysis

and in tabulated values of R0.

Page 7: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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

Förster radius - examples

Page 8: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

ETiiET

ET

kkk

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.

Energy transfer efficiency (E)

D

DA1

ED

DA1II

E or

Page 9: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

The distance dependence of the energy transfer efficiency (E)

0

61

11

RE

R

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.

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

0

0.25

0.5

0.75

1

0 20 40 60 80 100

Distance in Angstrom

Eff

icie

nc

y o

f tr

an

sfe

r

Page 10: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Homo energy transfer

Energy transfer between molecules of the same fluorophore

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

fluorescein

Page 11: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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

Page 12: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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

DA

D

1PB

PBE

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)

Page 13: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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

Page 14: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

FRET between a donor and acceptor, each attached to a

different protein, reports protein–protein interaction.

FRET concepts in protein science

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).

Page 15: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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

Page 16: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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 -helix inside the ß-barrel and a heterocyclic chromophor

Page 17: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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

Page 18: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Formation of the Chromophore

• formed by AS 65-67: Ser-Tyr-Gly

• p-Hydroxyben-zyliden-imidazolidon

• Isolated chromophore is not fluorescent

Y

S

Gfolding

cyclization

-H20

Page 19: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Fluorescence and Absorption (Wild-Type)

• Abs. at 395 and 475 nm

• Fluorescence at 509 nm

• Fluorescence almost

independent on ex

340 360 380 400 420 440 460 480 500 520 540 560 580 6000,0

0,2

0,4

0,6

0,8

1,0

Inte

nsi

ty

[nm]

Page 20: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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

Page 21: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

• Class 1: Wild–type

• Class 2: Phenol-anion

• Class 3: neutral Phenol in S0

Mutations lead to 6 classes of FP

EGFP

t203i

Page 22: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

• Class 4 : - interaction yellow emission

• Class 5 : Trp replaces Phenol cyan em.

• Class 3: Imidazole replaces Trp blue em.

Mutations lead to 6 classes of FP

YFP

CFP

BFP

Page 23: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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.

Truong et al. Nat Struct Biol, 8: 1069

480 535

+ C a2 +

-C a2 +

donor excitation at 440 nm

Fluorescence emission in nm

CFP YFP

E1

Page 24: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

1. 2 mM (Argon)2. 2 mM (air)3. 0.5 mM (Argon)4. 2 M (Argon – to

remove oxygen)

Excimer fluorescenceExcimer 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)*

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)

Page 25: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Application: An assay for membrane fusion. Fusion results in dilution of pyrene in the membrane → decrease in the

ration of excimer/monomer fluorescence intensity

E2Pyrene excimer fluorescence

Page 26: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Pyrene excimer formation can be used to determine diffusion coefficient (D) of lipids in bilayers of vesicles (using lipids labelled with pyrene)

Pyrene excimer and lipid diffusion in bilayers

M* + M → (MM)*kE kE = 2 D

cM – monomer surface concentration

Excimer formation competes with fluorescence of monomer and non-radiative deexcitation of monomer

monomer fluorescence:

excimer fluorescence:

MfMEnrf

fM kn

ckkkk

nI **

EEf

MEnrf

MEEnr

Ef

Ef

EE kckkk

ckn

kkk

nI

*

f

Ef

EMEM

E

kk

ckII

Determination of D via kE

constant for a fluorophore, ≈ 10 for pyrene

Page 27: Förster Energy Transfer Excimer Fluorescence Fluorescent Proteins Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

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


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