fluorescence quenching, fluorescence resonance energy...
TRANSCRIPT
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!