Transcript Fluorescence Spectroscopy

Fluorescence Spectroscopy
Part II. Quenching Techniques
Quenching
• Quenching
any process which reduces the lifetime of
the excited state.
reduction in the lifetime usually implies a
decrease in the quantum yield
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
Quencher
In protein study
• Iodide.
its charged nature quenches surface residues efficiently.
• Oxygen.
Small and can penetrate the protein to some degree.
• Acrylamide, nitroxides. neutral.
Mechanism of Quenching
Collisional or dynamic quenching
Static quenching
Quenching by energy transfer
Charge transfer reactions
Dynamic Quenching
When quenching occurs by a collisional mechanism, the quenching is
an additional process that deactivates the excited state besides
radiative emission.
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
F0
 1  K SV quencher
F
Stern-Volmer Relation
F0
 1  K SV quencher
F
•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.
Stern-Volmer Plot
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.
•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.
F
F*
hexc
F*
kr
F *+Q
F*


0
kQ[Q]
kIC
kr
F + hem
Fluorescence
F + Q Quenching
F + heat
Internal
conversion
k r  k IC  kQ [Q]
kr
k r  k IC
without quencher
kQ
0
I 0 t0
 1
[Q]  1  t0 kQ [Q]  

kr  kIC
I t
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.
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
Example
3 Trp in Chymosin
Example
3 Trp in Chymosin
Example 4
Interaction of Tritrpticin with micelle
Example 4
Interaction of
Tritrpticin with
micelle
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
I0
 1  K SV QeV Q
I
KSV
V
dynamic quenching constant
static quenching constant
Static Quenching
Example somatostatin in reverse micelle
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
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.
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.
Steady State Emission Anisotropy
Polarization Spectra
Relationship between polarization and rotational correlation time
1P 13   1  3 t 
 
 1   1 
t 
 P
3


0
0
t rot
4r 3 3V


kT
RT
P0 is the maximum P when the
rotational motion is very slow
trot rotational correlation time

solution viscosity
V= 4/3 r3N0= molar volume
Resonance Energy Transfer
• 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.
Emission of D quenched
Emission of A sensitized
• 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.
Resonance Energy Transfer
• FÖrster Theory
 1  R0 
 
kT  
 t D  R 
6
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
E  kT /(kT  1/ t D ) 
6
R0
6
/(R0
R )
6
Fluorescence Spectroscopy
Part III. Time-Resolved Fluorescence
Measurements
Time-Resolved
Fluorescence Spectra
Example Cutinase
20 oC
I F t   A  B exp k r t   C exp k d t 
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
Pulse Fluorimtetry
For single exponential decay
I t    exp  t / t 
For multi-exponential decay
I t  
N
 exp t / t 
i
i 1
i
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)
Quality of the fit
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.
Decay Curve
Decay of the fluorescence intensity (348.8 nm)after
pulse excitation (302 nm) of 10mM cutinase
Fresh
Irradiated
Example
Conformations and Orientations of Aromatic
Amino Acid Residues of Tachyplesin I in
Phospholipids Membranes
In the absence of liposomes
In the presence of liposomes
Tachyplesin I
[Phe8]-Tachyplesin I
[Phe13]-Tachyplesin I
Excitation wavelength 295 nm
in buffer
in liposomes
•Since tachyplesins are small the fluorescence anisotropy are small
in buffer solution
•In membrane, large increases in anisotropy values were observed
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
Fluorescence decay function
I  , t  
3
  exp t / t 
i
i
i 1
Associated Emission spectra
with individual decay time
component (DAS)
I i    I ss f i
f i   i  t i /
   t 
i
i
In Tris buffer
In neutral DMPC
In acidic DMPC
Example
Solvent-Exposed Tryptophans Probe the
dynamics at Protein Surfaces
Subtilisin Carlsberg
Subtilisin Carlsberg
Myelin basic protein