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

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!

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

How to plot the results of the quenching measurement?

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

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.

How can we decide the type of the quenching?

Dynamic quenching Static quenching

Application of quenching

• Membrane permeability

• Determination of diffusion constant

• Investigation of conformational states of proteins

Quenching of tryptophan with acryamide

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?

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.

• 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)

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.

+

-

FRET

+

-

+

-

D A E ~ kt ~ 1/R6

R

hν

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.

Molecular mechanism of FRET

• 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

66

0

6

0

RR

RE

Distance dependence of FRET

Förster distance in energy resonance transfer

distance between the fluorophores

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

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)

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.

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

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:

The fluorescence intensity of the donor

2.

10740400

The fluorescence intensity of the donor and the acceptor

4738510

E = 1 – (FDA / FD)

Calculation of the FRET efficiency

FDA=4738510

FD =10740400

E=55,88%

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.

How to calculate the Förster critical distance?

The emission spectrum of the

donor (tryptophan)

The absorption spectrum of the acceptor (IAEDANS)

Calculate the distance between the fluorophores!

R0=2,473 nm

R=2,158 nm

Application of FRET

• Measuring distance (molecular tape)

• Conformation of proteins

• Interaction of proteins

• Dissociation of macromolecules (pl. DNA)

Investigation of protein-protein interaction

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

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).

Thank you for your attention!