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Page 1: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

Fluorescence quenching, Fluorescence Resonance energy transfer (FRET)

Biophysics seminar

29. 02. 2012. http://en.wikipedia.org/wiki/Quenching_%28fluorescence%29 http://www.olympusmicro.com/primer/techniques/fluorescence/fret/fretintro.html

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Fluorescence quenching

• The decrease of the fluorescence intensity by

molecules able to interact with the fluorofor.

• Quencher is a special molecule which is

responsible for the quenching. It can take away the

excited energy of the fluorofor.

• The quenching process competes with the

fluorescence emission.

Decrease of the fluorescence intensity!

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Static quenching

• Due to the formation of dark complex between the ground state fluorofor and the quencher some of the fluorofor behave as a non-fluorescence molecule.

• Decrease of fluorescence intensity!

• Lifetime is not sensitive for the static quenching!

Types of fluorescence quenching

+ : + : + Quencher Fluorofor

Collision complex

(weak komplex)

h*υ

No emission!

Dark complex

(strong complex)

Excitation

Fluorofor Quencher

h*υ

Dynamic quenching • Due to the collision between the excited

state fluorofor and the quencher some of the fluorofor become de-excited.

• Diffusion controlled process. • Decrease of fluorescence intensity and

lifetime!

Excitation

Quencher Fluorofor

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How to plot the results of the quenching measurement?

The slope of the straight line gives the Stern-Volmer constant (KSV)!

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Stern-Volmer equation

Otto Stern (1888-1969)

Physical Nobel Prize (1943) Max Volmer (1885-1965)

F0 : Fluorescence intensity without quencher

F : Fluorescence intensity with quencher

Ksv : Stern-Volmer constant

[Q] : Concentration of quencher

][10 QKF

Fsv

00 SV

D

1 k q 1 K q

Inform about the accessibility of the fluorofor!

kq : bimolecular rate constant, informs about the diffusion

ability of the fluorofor and quencher, the accessibility of

the fluorofor

Ksv=kq* τ0

Stern-Volmer constant(Ksv)

Dynamic quenching

If the lifetime of the fluorophor is analysed as the funtion of quencher concentration, the result will be linear correlation.

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How can we decide the type of the quenching?

Dynamic quenching Static quenching

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Application of quenching

• Membrane permeability

• Determination of diffusion constant

• Investigation of conformational states of proteins

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Quenching of tryptophan with acryamide

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The access of the fluorophor Actin monomer

Nucleotide binding side

Cofilin Profilin

ε-ATP ε-ATP

How does the binding side change in the presence of an actin binding protein?

Page 10: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

0,0 0,1 0,2 0,3 0,4 0,5

1,00

1,25

1,50

1,75

2,00

2,25

2,50

2,75

3,00

= 20% (bound -ATP)

Ksv2 = 53.6 M

-1 (free -ATP)

Ksv1 = 0.28 M

-1

Ksvcofilin

= 0.09 M-1

Ksvprofilin

= 1.02 M-1

F0 /

F

Acrylamide concentration (M)

The nucelotide binding cleft shifted into an open conformation in the presence of profilin.

The nucelotide binding cleft shifted into a closed conformation in the presence of cofilin.

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• Perrin suggested that energy could be transferred over distances longer

than the molecular diameters.

• Theodor Förster developed the theoretical basis of FRET (1946).

• Förster type energy transfer: energy transfer between a donor and an

acceptor molecule through dipole-dipole interactions.

• Radiationless: photons are not involved.

• Fluorescence Resonance Energy Transfer (FRET): energy transfer

between fluorophores.

Fluorescence Resonance Energy Transfer (FRET)

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Dipole

• Apolar molecule: uniform charge distribution.

• Polar molecule: non-uniform charge distribution (the

positively and negatively charged parts are separated).

→ Dipole-molecule: polar molecule with two poles.

+

-

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FRET

+

-

+

-

D A E ~ kt ~ 1/R6

R

hν hν

FRET is a special type of quenching of the fluorescence which can decrease the fluorescence intensity of a given fluorophore (donor).

Donor: the source of the fluorescence.

Acceptor: a fluorophore that can absorb the energy accumulated in

the donor molecule.

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Molecular mechanism of FRET

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• Fluorescent donor and acceptor molecule.

• The distance (R) between the donor and acceptor molecule 2-10 nm! •Appropriate orientation of the dipoles of the fluorofores

• Overlap between the donor’s emission spectrum and the acceptor’s absorption spectrum.

wavelength (nm)

Fl in

tensity

and O

D

Conditions has to be fulfilled

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66

0

6

0

RR

RE

Distance dependence of FRET

Förster distance in energy resonance transfer

distance between the fluorophores

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0

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10 12 14 16 18 20

R (nm)

E

!!!!!!!22

0

2

0

RR

RE

66

0

6

0

RR

RE

Distance dependence of FRET

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How to determine the efficiency of FRET?

1. Time-dependent measurements (fluorescence lifetime)

E = 1 – ( DA / D)

2. „steady-state” measurements

E = 1 – (FDA / FD)

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0 20 40 60 80 100

1

10

100

1000

10000

100000

Inte

ns

ity

(c

ps

)

Time Domain Time (ns)

Intensity

IRF

Fit

The lifetime of the donor 1.

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The lifetime of donor and acceptor

0 20 40 60 80 100

1

10

100

1000

10000

Inte

ns

ity

(c

ps

)

Time Domain Time (ns)

Intensity

IRF

Fit

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Calculation of FRET efficiency

In case of lifetime measurement

E = 1 – (τDA / τD)

τD=2.959 ns τDA=1.191 ns

E=59.8%

Average lifetimes:

Page 22: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

The fluorescence intensity of the donor

2.

10740400

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The fluorescence intensity of the donor and the acceptor

4738510

Page 24: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

E = 1 – (FDA / FD)

Calculation of the FRET efficiency

FDA=4738510

FD =10740400

E=55,88%

Page 25: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

66

0

6

0

RR

RE

How to calculate the distance between the fluorophores?

Förster critical distance (R0):

Distance between the donor and acceptor where the

energy transfer is half of the maximum.

Page 26: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

How to calculate the Förster critical distance?

Page 27: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

The emission spectrum of the

donor (tryptophan)

The absorption spectrum of the acceptor (IAEDANS)

Page 28: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

Calculate the distance between the fluorophores!

R0=2,473 nm

R=2,158 nm

Page 29: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

Application of FRET

• Measuring distance (molecular tape)

• Conformation of proteins

• Interaction of proteins

• Dissociation of macromolecules (pl. DNA)

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Investigation of protein-protein interaction

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706461/?tool=pmcentrez#supplementary-material-sec

Page 32: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

Movies show two examples of HeLa cells microinjected with 10 ng/μg Cyclin A-Cer and

Cdk1YFP with images taken every 4 minutes starting 2hours after microinjection. Images are

overlays of the DIC image (greyscale) and the acceptor-normallized FRET efficiency (colour).

Page 33: Fluorescence quenching, Fluorescence Resonance energy ...biofizika2.aok.pte.hu/tantargyak/files/seminar2/... · Fluorescence quenching • The decrease of the fluorescence intensity

Thank you for your attention!


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