Transcript Fluorescence Spectroscopy

Fluorescence Spectroscopy
Part I. Background
Perrin-Jablonski
diagram
S is singlet and T is triplet.
The S0 state is the ground state and the subscript
numbers identify individual states.
Energy level of MO
n  p* < p  p* < n  s* < s  p* < s  s*
Singlet & Triplet
DS0
Characteristics of Excited States
Energy
Lifetime
Quantum Yield
Polarization
Stokes shift
The Stokes shift is the gap between the maximum of
the first absorption band and the maximum of the
fluorescence spectrum
loss of vibrational energy in the excited
state as heat by collision with solvent
heat
Example: 7-amino-4-methylcoumarin (AMC)
Example
Example
fluorophores
fluorescein
ethidium bromide
bound to DNA.
Lifetime
Lifetime
Excited states decay exponentially with time
– I = I0e-t/t
• I0 is the initial intensity at time zero,
• I is the intensity at some later time t
• t is the lifetime of the excited state.
• kF = 1/ t, where kF is the rate constant for fluorescence.
Quantum Yield
Quantum Yield = FF
• FF = number of fluorescence quanta emitted divided by
number of quanta absorbed to a singlet excited state
• FF = ratio of photons emitted to photons absorbed
•Quantum yield is the ratio of photons emitted to
photons absorbed by the system:
Quantum Yield
Quantum Yield & Structure rigidity
Polarization
• Molecule of interest is randomly oriented in a rigid matrix
(organic solvent at low temperature or room temperature
polymer). And plane polarized light is used as the
excitation source.
• Degree of polarization is defined as P
I|| and I^ are the intensities of the
observed parallel and perpendicular
components,
a is the angle between thee mission
and absorption transition moments.
If a is 0° than P = +1/2,
and if a is 90° than P = -1/3.
Experimental Measurements
• Steady-state measurements: F, I
• Time-Resolved measurements: t
Instruments
Inner Filter Effect
• At low concentration the emission of light is uniform
from the front to the back of sample cuvette.
• At high concentration more light is emitted from the
front than theback.
• Since emitted light only from the middle of the
cuvette is detected the concentration must be low to
assure accurate FF measurements.
Inner Filter Effect
Measurement of fluorescence quantum yields
fraction of intensity emitted at that
particular wavelength
fraction of total fluorescence
that is detected
If (em) = IAbs (ex). f . f(em). K
em
em
I0(ex)
em
em
fluorescence quantum yield
absorbed intensity at ex
measured intensity of
fluorescence at em
If A0
 I
f

I abs (ex )  I 0 (ex ) 1 10 A(ex )

I abs ex   2.303.Aex .I 0 ex 
 I abs (ex ). f .K  2.303 . A(ex ). I 0 (ex ). f .K
em
If we measure the sample and a standard under the
same experimental conditions, keeping ex constant:
Standards:
Quinine sulfate in H2SO4 1N: f =0.55
Fluorescein in NaOH 0.1N: f =0.93
sample
I
 f
em
standard
I
 f
Asample ex   f
 standard
. standard
ex   f
A
sample
em
Important : the index of refraction of the two solvents
(sample and standard) must be the same
Measurement of fluorescence lifetimes
pulsed source
Start
PMT
.
.
.
.
#events
different excitation flashes
The TCSPC measurement relies on the concept that
the probability distribution for emission of a single
photon after an excitation yields the actual intensity
against time distribution of all the photons emitted as a
result of the excitation. By sampling the single photon
emission after a large number of excitation flashes, the
experiment constructs this probability distribution.
t (nsec)
exc.
monochromator
sample
emission
monochromator
Time correlated single photon counting:
Dt
Stop
PMT
Intrinsic Fluorescence of Proteins and Peptides
Absorption
Fluorescence
Lifetime
Wavelength
Wavelength
ns
Absorptivity
Quantum
nm
nm
Tryptophan
2.6
280
5,600
348
0.20
Tyrosine
3.6
274
1,400
303
0.14
Phenylalanine
6.4
257
200
282
0.04
Tryptophan
•Tryptophan, the dominant intrinsic fluorophore, is generally
present at about 1mol% in proteins. A protein may possess just
one or a few Trp residues, which facilitates interpretation of the
spectral data.
•Tryptophan is very sensitive to its local environment. It is
possible to see changes in emission spectra in response to
conformational changes, subunit association, substrate binding,
denaturation, and anything that affects the local environment
surronding the indole ring. Also, Trp appears to be uniquely
sensitive to collisional quenching, either by externally added
quenchers, or by nearby groups in the protein.
•Tryptophan fluorescence can be selectively excited at 295-305
nm. (to avoid excitation of Tyr)
Example: Tyrosine and its derivatives
I
V
II
III
IV
II
I
III
V
IV
I
V
III
IV
II
• The position and structure of the
fluorescence suggests that the
indole residue is located in a
completely nonpolar region of the
protein. These results agree with Xray studies, which show that the
indole group is located in the
hydrophobic core of the protein.
• In the presence of a denaturing
agent, the TrpP emission loses its
structure and shifts to 351nm,
characteristic of a fully exposed Trp
residue.
Changes in emission spectra can
be used to follow protein unfolding
Emission spectra of Pseudomonas fluorescens
azurin Pfl.
For 275-nm excitation, a peak is observed due to th
tyrosine residue(s)
Example Time-resolved protein fluorescence
Fluorescence intensity (A.U.)
120
t2=5ns
100
80
t1=2ns
60
40
20
0
500
550
600
650
700
750
wavelength (nm)
1.2
Fluorescence intensity (A.U.)
Resolution of the contributions of individual
tryptophan residues in multi-tryptophan proteins.
I(,t)=ai()exp(-t/ti)
t1=2ns, t2= 5ns
1
0.8
0.6
 em
0.4
0.2
0
0
5
10
15
t (ns)
20
Green fluorescent protein (abbreviated GFP
Isolated from the Pacific jellyfish
Aequorea victoria and now plays
central roles in biochemistry and
cell biology due to its widespread
use as an in vivo reporter of gene
expression, cell lineage, protein
protein interactions and protein
trafficking
One of the most important attributes
of GFP which makes it so useful in
the life sciences is that the
luminescent chromophore is formed
in vivo, and can thus generate a
labeled cellular macromolecule
without the difficulties of labeling
with exogenous agents.
The structure of GFP : eleven-strand beta-barrel
wrapped around a central alpha-helix core. This
central core contains the chromophore which is
spontaneously formed from a chemical reaction
involving residues Ser 65, Tyr 66, and Gly 67
(SYG)
There is cyclization of the polypeptide backbone
between Ser 65 and Gly 67 to form a 5-membered
ring, followed by oxidation of Tyr 66.
The high quantum yield of GFP fluorescence
probably arises from the nearly complete
protection of the fluorophore from quenching
water or oxygen molecules by burial within the
beta-barrel.
Ribbon diagram of the
Green Fluorescent Protein
(GFP) drawn from the wildtype crystal structure. The
buried chromophore, which
is responsible for GFP's
luminescence, is shown in
full atomic detail.
Wild type GFP from jellyfish has two excitation peaks, a major one at 395 nm and a
minor one at 475 nm with extinction coefficient of 30,000 and 7,000 M-1 cm-1,
respectively. Its emission peak is at 509 nm in the lower green portion of the visible
spectrum.
For wild type GFP, exciting the protein at 395 nm leads to rapid quenching of the
fluorescence with an increase in the 475 nm excitation band. This photoisomerization
effect is prominent with irradiation of GFP by UV light. In a wide range of pH,
increasing pH leads to a reduction in fluorescence by 395 nm excitation and an
increased sensitivity to 475 nm excitation.
Melittin
GIGAVLKVLT TGLPALISWI KRKRQQX
Example
Carboxyfluorescence
Biochemical Education 28 (2000)
171~173
Example
Carboxyfluorescence
Quenching Effect
Example
Carboxyfluorescence
pH Effect