Fluorescence quenching, Fluorescence Resonance energy ... Fluorescence quenching ¢â‚¬¢ The decrease of

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  • 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 QK F

    F sv

    0 0 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)

    Ksv 2 = 53.6 M

    -1 (free -ATP)

    Ksv 1 = 0.28 M

    -1

    Ksv cofilin

    = 0.09 M -1

    Ksv profilin

    = 1.02 M -1

    F 0 /

    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

    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)

    F l in

    te n s it y

    a n d O

    D

    Conditions has to be fulfilled

  • 66

    0

    6

    0

    RR

    R E

    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

    R E

    66

    0

    6

    0

    RR

    R E

    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

    In te

    n s

    it y

    ( c

    p s

    )

    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

    In te

    n s

    it y

    ( c

    p s

    )

    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

    R E

    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!