Fluorescence quenching, Fluorescence anisotropy, Fluorescence resonance energy transfer (FRET)

Transcription

1 Fluorescence quenching, Fluorescence anisotropy, Fluorescence resonance energy transfer (FRET) Timescale of fluorescence processes The excited electron decay possibilities k f k ph k q k t k ic Biophysics seminar Dept. of Biophysics, University of Pécs Tamás Huber

2 Fluorescence quenching Quantum yield: number of the emitted photons divided by the number of the excited photons Lifetime: 63 % of the originally excited photons are already emitted the energy 1 Q f k f k nr k f kf k nr The decrease of the fluorescence intensity by molecules able to interact with the fluorophore. Quencher is a special molecule which is responsible for the quenching. It can take away the excited energy of the fluorophore. The quenching process competes with the fluorescence emission. Decrease of the fluorescence intensity! 2

3 Example 1. Static quenching Due to the formation of dark complex between the ground state fluorophore and the quencher some of the fluorophore behave as a non-fluorescence molecule. Is not affected by diffusion Lifetime is not sensitive for the static quenching Two samples of quinine dissolved in water with a violet laser (left) illuminating both. Typically quinine fluoresces blue, visible in the right sample. The left sample contains chloride ions which quenches quinine's fluorescence, so the left sample does not fluoresce visibly (the violet light is just refracted laser light). Fluorophore + : No emission Quencher Dark complex h*υ (strong complex) Excitation 3

4 Intensity Dynamic quenching Due to the collision between the excited state fluorophore and the quencher some of the fluorophore become de-excited. Diffusion controlled process Decrease of fluorescence intensity and lifetime! + : + Fluorofophore Quencher h*υ Collision complex (weak komplex) Fluorophore Quencher Concentration of quencher Excitation 4

5 If the lifetime of the fluorophor is analysed as the funtion of quencher concentration, the result will be linear correlation. 0 1 k 0 q 1 KSV q Stern-Volmer constant (K sv ): Informs about the accessibility of the fluorophor The slope of the straight line gives the Stern-Volmer constant (K SV )! Max Volmer ( ) Dynamic quenching K sv =k q * τ 0 k q : bimolecular rate constant, informs about the diffusion ability of the fluorophor and quencher, the accessibility of the fluorophor Otto Stern ( ) Physical Nobel Prize (1943) 5

6 F 0 : Fluorescence intensity without quencher F : Fluorescence intensity with quencher K sv : Stern-Volmer constant F 0 1 K [ Q sv ] F [Q] : Concentration of quencher More than one population of the fluorophores with different accessibilities to the quenchers. α : the fraction of the initial fluorescence which is accessible to the quencher 6

7 1. Neutral: acrylamid, nitroxis maping steric behavior 2. Charged: iodide, cesium, cobalt maping the charged surroundings Dinamic quenching Static quenching 7

8 Membrane permeability Determination of diffusion constant Investigation of conformational states of proteins 8

9 Polarized light is used in anisotropy measurements Absorption vector shows the direction in which the fluorophore prefer to absorb light If a fluorescent molecule is excited with polarized light the emission will also be polarized. The extent of polarization of the emission is usually described in terms of anisotropy (r). The emission vector shows the direction in which the fluorophor prefers to emit. 9

10 Tumbling of molecules the emitted light is depolarized. From a random organized population select a subpopulation, which absorption vector is parallel to the electric vector of the excitation light. The depolarization of the fluorescent molecule is dependent on: size shape of the rotating molecule viscosity of solution The smaller molecule The more the light is depolarized more rapidly rotation lower anisotropy If a larger molecule interacts with the fluorescent molecule the rotation of the complex will be slower than of the unbound molecules and result in an increase in the fluorescence anisotropy. 10

11 excitation I x x z I z E I y y emission vector intensity Detector I VV The observed intensity when the emission polarizer is oriented parallel to the direction of the polarized excitation. I VH - The observed intensity when polarizer is perpendicular to the excitation. G G factor, correction factor G=I HV /I HH, the sensitivity of the detector and the monochromator is not the same for the vertically and horizontally polarized light. v Emission anisotropy: Difference of intensity vectors normalized to the total intensity Anisotropy of different fluorophores is additive r I I VV VV GI 2GI VH VH Perrin equation r 1 r r 1 6D 0 0 Stokes Einstein relationship V RT V could be determined r 0 : limiting anisotropy ( frozen molecule, if r = r 0 φ infinite) τ: fluorescence lifetime φ: rotational correlation time ( flexibility ) D: diffusion coefficient V: fluorescent volume η: viscosity of the medium T: absolute temperature R: universal gas constant 11

12 Application of anisotropy Determination of rotation diffusion Detection of intermolecular interaction Observation of conformational changing Non-radiative dipole-dipole interaction between a donor and an acceptor fluorophore. The donor fluorophore gives the excited state energy to the acceptor fluorophore. Donor Acceptor k ex k nf k f k t k f Theodor Förster

13 Absorbance Fluorescence intensity Donor and acceptor fluorophores Distance range between 2-10nm Appropriate orientation of the dipoles of the fluorofores Spectral overlap between the emission of the donor and the absorption of the acceptor τ DA : lifetime of donor in the presence of acceptor τ D : lifetime of donor in the absance of acceptor F DA : fluorescence intensity of donor in the presence of acceptor F D : fluorescence intensity of donor in the absance of acceptor Decrease of intensity of donor! k t = const. * J(λ) n -4 k f R -6 κ 2 Increase of intensity of acceptor Energy transfer Wavelength (nm) 13

14 E R 6 0 R 6 0 R 6 Förster critical distance (R 0 ): Distance between the donor and acceptor where the energy transfer is half of the maximum. 14

15 Heterotransfer: between different fluorophore Homotransfer: between identical fluorophore Homotransfer Heterotransfer 15

16 Nucleotide binding site Nucleotide binding site εatp εatp 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. 16

17 Measuring distance (molecular ruler) Conformation of proteins Interaction of proteins Dissociation of macromolecules (e.g. DNA) 17

18 Intensity (cps) Intensity (cps) Intensity IRF Fit Intensity IRF Fit Time Domain Time (ns) Time Domain Time (ns) 18

19 2. In case of lifetime measurement E = 1 (τ DA / τ D ) Average lifetimes: τ D =2.959 ns τ DA =1.191 ns E=59.8% 19

20 E = 1 (F DA / F D ) F DA = F D = E=55.88% 20

21 Thank you for your attention! 21