1. Fluorescence Spectroscopy
Part II. Quenching Techniques

2. Quenching
Quenching
any process which reduces the lifetime of the excited state.
reduction in the lifetime usually implies a decrease in the quantum yield

3. Quencher
Collisional and static quenching require contact between the fluorophore and the quenching. Thus, these methods are useful to measure rates of diffusion and exposure of fluorescent species to the quencher.
A large number of quenchers are known and a partial list is: molecular oxygen, amides, BrO4-, xenon, I-, peroxides, nitroxides, acrylamide

4. Quencher In protein study Iodide. Oxygen.
its charged nature quenches surface residues efficiently.
Oxygen.
Small and can penetrate the protein to some degree.
Acrylamide, nitroxides. neutral.

5. Mechanism of Quenching
Collisional or dynamic quenching
Static quenching
Quenching by energy transfer
Charge transfer reactions

6. Dynamic Quenching
When quenching occurs by a collisional mechanism, the quenching is an additional process that deactivates the excited state besides radiative emission.

7. Stern-Volmer Relation
FF fluorescent intensity in the absence of quencher
FFQ fluorescent intensity in the presence of quencher
t fluorophore decay lifetime in the absence of Q
kq the quenching constant
Let KSV = kqt
Stern-Volmer Equation

8. Stern-Volmer Relation
Since KSV = kqt if t is known, then kq values can be obtained from the slope of a plot of F0/F vs. [quencher].
If quenching results from a nonfluorescent ground state complex between the fluorophore and the quencher, the dependence of F0/F on [quencher] is identical to that observed for dynamic quenching except the quenching constant is now the association constant.

9. Stern-Volmer Plot

11. Application of quenching Techniques
study of proteins is to determine the location of Trp residues
change in location of Trp residues, due to conformational changes.
Reduction in the quenching of Trp residues bound to proteins versus that of free Trp is observed, and this can arise from two factors.
quenching of Trp is reduced to that of free Trp (or indole) because the Trp is now attached to a molecule with a smaller diffusion co-efficient. For proteins greater than 50 KDa in size, this results in a decrease in the quenching constant of 50.
Trp residue is buried in the protein and is thus not accessible to quenching agent unless the quenching agent can diffuse into the protein interior.
The correlation between acrylamide quenching and the emission wavelength of the Trp residue suggests that Trp residues buried in hydrophobic regions of the protein are less accessible for quenching.

12. The accessible fluorophores experience a decrease in fluorescence upon collision with collisional quenchers.
Charge versus steric effect is differentiated by quenchers with different size and charges.
Accessibility depends on exposure as well as lifetime.
Accessdibility of residues are reflected in the Stern-Volmer constant
For single tryptophan containing proteins, low values of kq indicate residues of low exposure, ie those buried within the protein structure.
These residues also have blue shifted emissions indicating a relatively nonpolar environment.

13. hexc F F* kr F * F + hem kQ[Q] F *+Q F + Q kIC F * F + heat
Fluorescence
kQ[Q]
F *+Q F + Q
Quenching
kIC
F * F + heat
Internal conversion
without quencher
Collisional quenching of proteins has been extensively utilized to determine the extent of Trp exposure to the aqueous phase.
Collisional quenching requires the fluorophore and quencher to be in molecular contact. A water-soluble quencher will not easily penetrate the protein matrix.
kQ reflects the efficiency of quenching, or the accessibility of the fluorophores to the quencher.
Diffusion-controlled quenching typically results in values of kQ near 1010 M-1s-1.

14. Example 1
Apoazurin Ade has two Trp residues, one surface residue and one buried residue.
In the presence of iodide (collisional quencher), the spectrum resembles the structured emission seen in non-polar environments.
The spectrum of the quenched Trp can be seen from the difference spectrum (dashed line), and is characteristic of an exposed residue in a partially hydrophobic environment.

15. Example 2 Accessibility of Trp Residues in Lysozyme
contains six tryptophan residues. positions 28, 62, 63, 108, 111 and 123.
portion of these residues exposed to the aqueous solvent can be determined by Stern-Volmer equation .
Fo = Fo,a + Fo,b
F = F o,a / (1+KSV[Q]) + F o,b
dF = Fo - F = Fo,aKSV[Q] / (1+KSV[Q])
Fo / (F0-F) = 1 / (KSV [Q]) + 1/fa
fa is the fraction of accessible fluorophores obtained from intercept
KSV can be obtained form the slope

16. Example 3 Trp in Chymosin

17. Example 3 Trp in Chymosin

18. Example 4 Interaction of Tritrpticin with micelle

19. Example 4 Interaction of Tritrpticin with micelle

20. Static Quenching
Sometimes Stern-Volmer plots show a positive deviation from linearity for the highest concentrations of quencher used.
These deviations are interpreted according to the sphere-of-action static quenching model.
If a quencher is within a volume of V’ around the fluorophore, a quenching reaction occurs instantaneously after excitation with unit efficiency.
Assuming that the quenchers are distributed among these volumes according to a Poisson distribution, the following expression for the combined existence of dynamic and static quenching is obtained
KSV dynamic quenching constant
V static quenching constant

21. Static Quenching

23. Example somatostatin in reverse micelle

24. Example somatostatin in reverse micelle
Stern-Volmer plots are non-linear and exhibit upward curvature
Only some fraction of the excited states is actually quenched by the usual collisional mechanism.
Some of the excited states are deactivated statically after being formed by either an active volume element surrounding the fluorophoreor a dark complex between the reactant

25. Example Fusarium solani Cutinase
In native state the Trp fluorescence is highly quenched. Irradiation of the enzyme in the Trp absorption band causes an increase of fluorescence quantum yield.

26. Steady State Emission Anisotropy Polarization Spectra
The definition of the emission anisotropy P in a configuration where the exciting light is vertically polarized and the emitted fluorescence is observed at right angles in a horizontal plane P
I|| and I are the polarized components parallel and perpendicular to the direction of polarization of the incident light, respectively.

27. Steady State Emission Anisotropy Polarization Spectra

28. Relationship between polarization and rotational correlation time
P0 is the maximum P when the rotational motion is very slow
trot rotational correlation time
h solution viscosity
V= 4/3 pr3N0= molar volume

29. Resonance Energy Transfer
Emission of D quenched
Emission of A sensitized
Energy transfer is the non-radiative transfer of energy from a donor to an acceptor. For energy transfer to occur the absorption spectra of the acceptor must overlap the emission spectra of the donor.
The overlap is required such that quantum energy levels of equivalent energy exist for productive dipolar coupling between the two molecules.
the rate of transfer depends on the distance between the donor and acceptor, energy transfer can be used to measure distances between sites on biopolymers. The distance range is much larger (20-70A) than possible with other spectroscopic techniques (e.g. NMR - 5 A), thus making fluorescence energy transfer a very useful technique.

30. Resonance Energy Transfer
FÖrster Theory
kT rate of energy transfer
tD life-time of D in the absence of A
R0 characteristic transfer distance~10-50 Å
Efficiency of Energy Transfer

31. Fluorescence Spectroscopy
Part III. Time-Resolved Fluorescence Measurements

32. Time-Resolved Fluorescence Spectra

33. Example Cutinase
20 oC
IF(t) intensity of fluorescence measured at time t
kr rate constant for the rise of the fluorescence intensity
kd rate constant for the decay of the fluorescence intensity
40 oC

35. Pulse Fluorimtetry For single exponential decay
For multi-exponential decay

36. Obtain Decay curve of the fluorescence intensity
after pulse excitation
Fit the experimental decay curve
With multi-exponential function
Judge the fit with
c (close to 1)
W (randomly distributed around 0)

37. Quality of the fit weighted residuals,
In the least-squares method, the first criterion is the reduced cr2 whose value should be close to 1 for a good fit.
weighted residuals,
If the fit is good, the weighted residuals are randomly distributed around zero.
Acceptable values are in the 0.8–1.2 range. Lower values indicate that the data set is too small for a meaningful fit and higher values are caused by a significant deviation from the theoretical model (e.g. insufficient number of exponential terms).
Systematic errors (arising for instance from radiofrequencies interfering with the detection) can also explain higher values.

38. Decay Curve

39. Decay of the fluorescence intensity (348
Decay of the fluorescence intensity (348.8 nm)after pulse excitation (302 nm) of 10mM cutinase
Fresh
Irradiated

40. Example
Conformations and Orientations of Aromatic Amino Acid Residues of Tachyplesin I in Phospholipids Membranes

41. In the absence of liposomes In the presence of liposomes

42. Tachyplesin I
[Phe8]-Tachyplesin I
[Phe13]-Tachyplesin I
Excitation wavelength 295 nm
in buffer
in liposomes

43. Since tachyplesins are small the fluorescence anisotropy are small in buffer solution
In membrane, large increases in anisotropy values were observed

44. Quality of the fluorescence decay analysis
Tachyplesin I in Tris buffer
Ex: 295 nm; em: 350 nm
One-exponential
Double-exponential
Triple-exponential
Quadruple-exponential

45. Fluorescence decay function
Associated Emission spectra with individual decay time component (DAS)

46. In Tris buffer
In neutral DMPC
In acidic DMPC

49. Solvent-Exposed Tryptophans Probe the dynamics at Protein Surfaces
Example
Solvent-Exposed Tryptophans Probe the dynamics at Protein Surfaces

50. Subtilisin Carlsberg

51. Subtilisin Carlsberg
Myelin basic protein