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Fluorescence methods in life sciences
Ctirad Hofr
Intrinsic protein
fluorescence
Examples of fluorescent animals
What does really shine
there?
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Fluorophores around us
Fluorophores are divided into two general classes:
intrinsic – they occur naturally e.g. in proteins
extrinsic – they are added to samples which do not have suitable fluorescence
properties
Protein fluorescence
Aromatic amino acids tryptophan (Trp), tyrosine (Tyr) a phenylalanine (Phe)
are major fluorophores in proteins. Their absorption band lies between 240 and
300 nm, emissions is also in the ultraviolet region. Dominant fluorophore is
tryptophan, respectively its indole group, because it has a much broader
emission spectrum than tyrosine, whose fluorescence is due to its phenol ring.
Phenylalanine practically does not contribute to the total fluorescence of
proteins. Tryptophan fluorescence is very sensitive to the properties of its
surroundings and therefore it can be used for the observation of protein
conformational changes (e.g. in ligand binding or protein-protein interactions).
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Absorption and emission spectrum
of aromatic amino acids
• Proteins are fluorescent due to three
aromatic amino acids:
tryptophan, tyrosine and phenylalanine
• In the sequence they occur relatively little
• Tryptophan (Trp) occurs only in 1% of
molecules, which is probably due to its
relatively metabolically demanding
synthesis
• Tryptophan is sensitive to the local
environment
fluorophor λex
max
(nm)
λem
max
(nm)
quantum
yield
Lifetime
(ns)
tryptophan 295 353 0,13 3,1
tyrosine 275 304 0,14 3,6
phenylalanine 260 282 0,02 6,8
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Change of emission spectrum
depending on the environment
Tryptophan emission is influenced by the
surrounding environment – by the local electric
field of protein, polarity of solvent and ionic
strength of solution
Polarity ionic strength
http://micro.magnet.fsu.edu/primer/java/jablonski/solventeffects/index.html
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Influence of the protein environment
on the emission spectrum of tryptophan
1 apoazurin PFl
2 ribonuclease T1
3 Staphylococcus
nuclease
4 glucagon
Tryptophan in a nonpolar
environment has emission
maximum shifted to the shortest
wavelength (apoazurin)
Tryptophan exposed to the polar
solution has a maximum at the
longest wavelength (glucagon)
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Factors influencing the tryptophan emission
• Quenching by proton transfer from close amino
groups
• Quenching by electron acceptors (COO-)
• Quenching by electron transfer from disulfides
and amides
• Quenching by electron transfer from peptide
bonds of protein backbone
• FRET between different molecules of tryptophan
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Emissions of tryptophan in protein
nonpolar environment - azurin
• Azurin from Pseudomonas aeruginasa is a
protein that contains a copper ion and is
involved in electron transfer of
denitrification bacteria
• Structure is composed of α-helix and
8 β-sheets, which form β-barel structure
with a hydrophobic core
• In the native state, the emission spectrum
is the same as in the case of a nonpolar
environment (e.g. cyclohexane)
• In the denatured state, the emission
spectrum is shifted, which correspond to
the movement of tryptophan into the polar
environment
• The emission spectrum is different at
different excitation wavelength, because
at 275 nm tyrosine is also excited (peak
~ 300 nm), while at 292 nm only
tryptophan is excited
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275 292
Absorption and emission spectrum
of aromatic amino acids
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Emissions of tryptophan when ambient
amino acids are exchanged
• When nonpolar isoleucine and
phenylalanine were replaced by
directed mutagenesis of the azurin
for polar serin, there was a shift of
the spectrum to longer wavelengths
• Substitution at serine with OH group
induces a similar effect as if Trp is in
ethanol
• Just a small change nearby Trp
caused a significant shift of spectrum
N
O
isoleucine
N
O
phenylalanine
N
O
O
serine
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FRET between amino acids
• Between different aromatic amino acids, a resonant energy transfer is
found
• FRET between tyrosine and tryptophan is most common because they
most often occur in protein sequence and they both are excited at 275 nm
• The rate of energy transfer is given by the emission spectrum and
quantum yield of amino acid which serves as a donor
• The rate of energy transfer is dependent on the surrounding environments
of individual amino acids
• The resonant transfer may also occur between two molecules of Trp in the
case that one is in a nonpolar environments and the second is exposed to
the external environment (polar solution)
Donor Akceptor R0 (A)
Phe Tyr 11-13
Tyr Tyr 9-16
Tyr Trp 9-18
Trp Trp 4-16
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FRET between tyrosine and
tryptophan for interferon γ
• Interferon γ is produced by activated lymphocytes and
has antiviral and immunoregulatory effects
• Interferon activity is dependent on a proportional
amounts of dimers
• Intrinsic fluorescence of interferon which is given by 4
tyrosines and + tryptophan was used for observation
of dissociation of dimers to monomers
• Emission spectrum of dimer at the excitation of 270 nm
shows that tryptophan and tyrosine contributes to
fluorescence
• Only tryptophan emission is observed at the excitation of
295 nm
• The difference in the emission spectra at excitation of
270 nm and 295 nm indicates the spectrum of tyrosine
• In the case of dimer, the relative emission intensity of
tyrosine is 20 % compared with the emission of
tryptophan
• In the case of monomer, the relative emission intensity
of tyrosine is 50 % compared with the emission of
tryptophan
• Increase of tyrosine relative intensity after dissociation
is given by tryptophans moving away (FRET acceptors)
which were in dimer in close proximity to tyrosines
• Reduction of energy transfer between tryptophan and
tyrosine showed that during dissociation four "external"
tyrosine residues are moved away from tryptophans
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Utilization of FRET between amino acids to
determine the spatial arrangement
• Membrane protein M13 is nested into the
membrane of E. coli when connecting
phage particles
• It was suggested that two α helices of this
protein are in the membrane in close
proximity
• Protein variants were created by directed
mutagenesis, these variants include
tyrosine and tryptophan (donor -akceptor
FRET) in different positions
• Only one pair tyrosine - tryptophan was
always kept
• Emission of fluorescence at excitation of
280 (---) a 295 nm (…) was observed and
from the difference tyrosine emission was
determined
• If the distance of tyrosine and tryptophan
is larger, the emission intensity of tyrosine
is higher
• If tyrosine and tryptophan are in close
proximity, FRET occurs, that significantly
reduces the emission of tyrosine
• Reduction of tyrosine emission intensity in
case of W39Y(-15) demonstrated that the
α helices are in close proximity
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Fluorescence quenching
• Fluorescence quenching can be defined as bimolecular process
that reduces the quantum yield of fluorescence (ie. fluorescence
intensity) without changing of fluorescence emission spectrum.
• Collision (dynamic) quenching occurs when the fluorophore in
excited state is deactivated (i.e. it returns to the ground state
without radiation) in collision with molecule of quencher. In this
process the molecules are not chemically modified in contrast to
• static quenching when non-fluorescent complex is formed after
contact the fluorophore and the quencher
• Self-quenching means quenching of the fluorophore by itself; it
occurs with high concentrations of fluorophore or with high density
of labeling.
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Dynamic quenching
Reduction of fluorescence intensity by dynamic quenching is described by
Stern-Volmer equation:
F0/F = τ0/τ = 1 + kq τ0 Cq
F0 – fluorescence intensity in the absence of quencher,
F - the same in the presence of quencher with concentration Cq, τ0 –
fluorescence lifetime without quencher, τ - lifetime in the presence of
quencher, kq – bimolecular quenching constant
(= bimolecular rate constant determined by diffusion multiplied by
efficiency of quenching).
kq value indicates the concentration of quencher at which the
fluorescence intensity is reduced to half.
The most effective dynamic quencher of fluorescence and
phosphorescence is molecular oxygen (O2). Furthermore, halogen atoms
such as bromine and iodine quench the fluorescence (as a result of
inter-system conversion). Acrylamide is also frequently used quencher.
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How is it possible, that oxygen gets into
internal regions of the protein?
• It is small and proteins „breathe"
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Static quenching
• Complex of fluorophore and quencher is formed which
already does not emit fluorescence
• The Stern-Volmer equation is also valid for it:
F0/F = 1 + Ka τ0 Cq
Ka is the association constant of the fluorophore and
quencher
No change of life time! (on/off state of fluorophore)
Typical static quenchers are:
Bases of nucleic acids
mainly Guanine
Nicotinamide
heavy metals
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Use of quenching
in fluorophore localization
In membrane
• If fluorophore P1 is buried into
membrane, it is unavailable for
quencher Q and there is almost
no quenching.
• With increasing concentration of
quencher the fluorescence
intensity is almost unchanged.
On the surface
• If fluorophore P2 is on the
surface the efficient quenching
occurs.
• With increasing concentration of
quencher the fluorescence
intensity decreases dramatically.
Burried in the
membrane
Exposed on the
surface
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Quenching in the study of protein structure
or
Is tryptophan on the surface of protein?
• Dynamic quenching
requires direct contact
of the fluorophore and
quencher
• When tryptophan is
located „inside“ protein,
its quenching does not
occur (W1)
• When tryptophan is
located on the surface
of protein, quenching
occurs (W2)
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Quenching in the study of apoazurin
• Apoazurin has two
tryptophans in the
sequence
• One tryptophan is on
the surface, the
second is buried
• After adding
quencher (KI) to the
solution, quenching
of tryptophan on the
surface occurs
• DS is a differential
spectrum
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Influence of protein environment on
tryptophan emission spectrum
1 apoazurin PFl
2 ribonuclease T1
3 Staphylococcus
nuclease
4 glucagon
Tryptophan in a nonpolar
environment has emission
maximum shifted to the shortest
wavelength (apoazurin)
Tryptophan exposed to the polar
solution has a maximum at the
longest wavelength (glucagon)
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Quenching of protein with one
tryptophan in acrylamide solution
• The higher slope of the Stern-Volmer graph, the greater degree of
quenching
• Tryptophan which is free in solution is the best quenched
(represented by NATA- N-acetyl-L-tryptophanamide)
• Tryptophan in azurin is the least accessible for quencher
NH
O
N
H O
NH2
Adrenocorticotropin
hormone
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Quenching of 2 tryptophans in myoglobin
During quenching of tryptophans from horse myoglobin (contains 2 Trp)
it was found that the proportional half of intensity is quenched.
This led to confirmation that tryptophan W14 is located in the inner part
of the structure between α helices, while W7 is on the protein surface
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Order determination of occupancy of
binding sites
• Calmodulin binds to calcium ions, its
structural change occurs, resulting in
increased ability of binding to receptors
and proteins, which metabolism regulates.
• One tryptophan was introduced always
close to one of the four calmodulin binding
sites and the change in fluorescence was
observed with increasing concentrations of
calcium ions
• Comparison of dependencies showed
order of occupancy of individual binding
sites by calcium cations
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Protein-DNA interaction
• During interaction of the protein with DNA, quenching of tryptophan by DNA bases occurs
• Protein SSB (single-stranded DNA binding) binds DNA with high affinity but low specifity
• Homotetramer is formed, that binds to DNA with a length of about 70 nucleotides
• Reduction of tryptophan emission after association of DNA and protein showed that there is a
DNA strand wrapping around tetramer
• Labelling of DNA at one end by donor and at the other end by acceptor showed DNA wraping
around protein complex
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Quenching of tryptophan fluorescence in
Myb oncoprotein during DNA binding
• Myb oncoprotein is associated with
chromatin and regulates gene
expression
• N-terminal part of the protein
contains R1, R2 a R3 domains
• Each domain contains three
tryptophans that are highly conserved
in sequence, suggesting that they are
involved in DNA binding
• Fluorescence of R2R3 fragment of
Myb oncoprotein is partially
quenched after binding to DNA
• After plotting of fluorescence
dependence on the concentration of
DNA, it was found that there was a
intensity quenching of 67%
• It follows that 2 of 3 tryptophans in
each domain are involved in the
interaction with DNA
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Protein with fluorescence in the
name
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Green Fluorescent Protein
• Composed of 238 amino
acids
• “Paint in a can”
• Monomer is composed of a
central α helix surrounded
by 11 antiparallel β chain in
the shape of cylinder
• Diameter of the structure is
30A and length is 40A
• Fluorophore is located on
the central α helix
http://www.cytographica.com/animations/FRET2.html
λ ex= 488 nm, λ em=507nm
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Fluorophore in GFP
• Ser65-Tyr66-Gly67
• Fluorophore is formed autocatalyticaly during
protein folding ( 2-4 hours)
• Tyr66 is a source of fluorescence
• The fluorophore was created by oxidation of
Tyr66 and subsequent spontaneous
cyclization
• The resulting fluorophore contains conjugated
double bonds
http://www.olympusconfocal.com/java/fpfluorophores/gfpfluorophore/inde
x.html
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Utilization of GFP
• Protein localization
• Dynamics of proteins and organelles
in vivo
• Transport between organelles
• Reporter of gene expression
• Flow cytometry
• Protein purification
• Movement of cells
GFP can be used to label proteins in living cells of various organisms due to
its inherent fluorescence (only gene coding GFP is needed – c-DNA 1992,
Prasher)
Why does scorpion emit fluorescence?
• Cuticula of scorpion contains
beta-Carboline (9H-pyrido[3,4-b]indole)
• It is a derivative of tryptophan, which is
formed after hardening of the
breastplate
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http://fireflyforest.net/firefly/2006/11
/13/fluorescent-scorpion-in-uv-light/
Stachel, Shawn J; Scott A Stockwell and David L Van Vranken (August
1999). "The fluorescence of scorpions and cataractogenesis".
Chemistry & Biology (Cell Press) 6: 531–539.
β carboline
Superstitionia donensis
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Literature
• Lakowicz J.R.: Principles of Fluorescence
Spectroscopy. Third Edition, Springer +
Business Media, New York, 2006.
• Fišar Z.: FLUORESCENČNÍ SPEKTROSKOPIE
V NEUROVĚDÁCH
http://www1.lf1.cuni.cz/~zfisar/fluorescence/Default.htm
Graphics from the book Principles of Fluorescence was for the purpose
of this lecture kindly provided by Professor JR Lakowitz.
Acknowledgment