Fluorescence

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Fluorescence

• Intrinsic Fluorescence Tryptophan
• Green Fluorescent Protein
• Quenching Mechanisms
• Fluorescent Energy Transfer
• Applications

NC State University
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Mirror image relationship between absorption and
fluorescence bands
Fluorescence
Absorption
Energy
0-1
0-2
1-0
2-0
0-3
3-0
0-0
4-0
0-4
Wavelength
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Rhodamine is an example of a mirror image
relationship
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Fluorescence lifetime and quantum yield
The intrinsic lifetime for a single state is
given by a single exponential with time constant
t0 The quantum yield is the ratio of the
molecules that decay by the fluorescent pathway
to the total
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Caspases
Caspases are cysteine proteases that are
activated during apoptosis. As with other
proteases they have an inactive form and an
active form. Splicing and folding of caspases
is required to initiate their function in
controlled cell death. Shown in the figure is
the structure of caspase-1. Note that the
structure indicates that two subunits have been
cleaved and are intermingled. This is the
active form.
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Tryptophan an intrinsic probe
The absorption is a p-p transition.
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Fluoresence of procaspase-3 (C163S)
Fluorescence
Fluorescence
280 nm excitation
295 nm excitation
Different spectra at 280 (aromatics) and 295
(tryptophan)
Dr. Clay Clark - Biochemistry
NC State University
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Unfolding of procaspase-3 (C163S)
Table 1.
Relative Fluorescence (320 nm)
Concentration dependence of folding curve is
shown. Urea denatures the protein. There is
clear evidence for two intermediates.
Dr. Clay Clark - Biochemistry
NC State University
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Refolding kinetics of procaspase-3 (C163S)
Relative Fluorescence
The protein is rapidly diluted and refolding is
observed on three different exponential time
scales.
The thermodynamic intermediates are shown
obtained from the equilibrium data.
Dr. Clay Clark - Biochemistry
NC State University
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Kinetic schemes
In a two state model there are no intermediates
kF
U unfolded F folded
F
U
kU
In a this case the equilibrium constant is
The time course for reaching equilibrium is
where kobs kF kU
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Kinetic schemes one intermediate
For one intermediate
U unfolded I intermediate F folded
k1
k2
I
F
U
k-1
k-2
In a this case the equilibrium constants are
However, the time course for reaching equilibrium
is biexponential. The kinetics depend on whether
U, I or F is being observed. However, as a
general rule the approach to equilibrium for N
intermediates involves N1 exponential rate
constants.
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GFP is formed by post-translational modification
Then, several chemical transformations occur the
glycine forms a chemical bond with the serine,
forming a new closed ring, which then
spontaneously dehydrates. Finally, over the
course of an hour or so, oxygen from the
surrounding environment attacks a bond in the
tyrosine, forming a new double bond and creating
the fluorescent chromophore. Since GFP makes its
own chromophore, it is perfect for genetic
engineering. You don't have to worry about
manipulating any strange chromophores you simply
engineer the cell with the genetic instructions
for building the GFP protein, and GFP folds up by
itself and starts to glow.
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Green Fluorescent Protein (GFP)
The green fluorescent protein is found in a
jellyfish that lives in the cold waters of the
north Pacific. The jellyfish contains a
bioluminescent protein-- aequorin--that emits
blue light. The green fluorescent protein
converts this light to green light, which is
what we actually see when the jellyfish lights
up. Solutions of purified GFP look yellow under
typical room lights, but when taken outdoors in
sunlight, they glow with a bright green color.
The protein absorbs ultraviolet light from the
sunlight, and then emits it as lower-energy
green light. GFP is useful in scientific
research, because it allows us to look directly
into the inner workings of cells. It is easy to
see where GFP is at any given time you just
have to shine UV light, and any GFP will glow
bright green. So the trick is to attach GFP to
any object that you are interested in watching.
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GFP Structure
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The cyclization reaction

• The amide N of Gly67 is in close enough contact
  to the carbonyl of Ser65 to generate a large
  conjugated resonant p-system capable of light
  absorption.
• The chromophore is held in place by several amino
  acids which form a tight hydrogen bond network
  including some fixed water molecules (distances
  for possible hydrogen bonds given in Å).

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The chromophore in 3D
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GFP as a reporter system

• Green fluorescent proten is particularly useful
  as a reporter in living cells and organisms. GFP
  gene fusions provide a "window" on to the
  mechanisms that regulate the activity of specific
  genes, in specific, living cells.
• As the message is transcribed for the gene of
  interest,
• GFP is made. Detection of GFP activity is a
• measure of gene expression.

Pol
Promotor
Green Fluorescent Protein
Gene of interest
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C. elegans expressing GFP alternative splicing
reporter constructs. It is now apparent from the
sequencing of the human genome that about half
of human genes are alternatively spliced and
that this alternative splicing is important for
the generation of the diversity of the human
proteome. Alternative splicing is regulated by
cis-acting sequences in the pre-mRNA, found
both in introns and exons. The Figure is from
the laboratory of Alan Zahler, UC Santa Cruz
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Fluorescence quenching

• The quantum yield gives the intrinsic fraction of
  the molecules
• that decay by a emitting light. In addition, the
  fluorescence
• emission can be further quenched by
• Collisional quenching molecular collisions in
  solution
• Intersystem crossing conversion from singlet to
  triplet
• Electron transfer 1DA ? DA-
• Energy transfer emission is transferred to an
  acceptor

The Stern-Volmer equation is F 0/F 1 kqt0
Q kq is the quenching rate constant, t0 is the
lifetime in the absence of quencher, Q is the
quencher concentration.
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Spectral overlap permits quenching
Spectral overlap between EDANS fluorescence and
DABCYL absorption permits efficient quenching
of EDANS fluorescence by resonance energy
transfer to nonfluorescent DABCYL.
Donor Acceptor
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Spectral overlap permits quenching
Spectral overlap between EDANS fluorescence and
DABCYL absorption permits efficient quenching
of EDANS fluorescence by resonance energy
transfer to nonfluorescent DABCYL.
hn
Donor Acceptor
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Spectral overlap permits quenching
Spectral overlap between EDANS fluorescence and
DABCYL absorption permits efficient quenching
of EDANS fluorescence by resonance energy
transfer to nonfluorescent DABCYL.
hn
Donor Acceptor
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Spectral overlap permits quenching
Spectral overlap between EDANS fluorescence and
DABCYL absorption permits efficient quenching
of EDANS fluorescence by resonance energy
transfer to nonfluorescent DABCYL.
Non-radiatiave
Donor Acceptor
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Spectral overlap permits quenching
Spectral overlap between EDANS fluorescence and
DABCYL absorption permits efficient quenching
of EDANS fluorescence by resonance energy
transfer to nonfluorescent DABCYL.
hn
Donor Acceptor
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Fluorophore
5-((2-aminoethyl)amino)naphthalene-1-sulfonic
acid, sodium salt (EDANS)
Quencher
e-(4-dimethylaminophenylazobenzoyl) -L-lysine
(DABCYL)
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Fluorescent resonant energy transfer (FRET)
Fluorescence resonance energy transfer (FRET) is
a distance-dependent interaction between the
electronic excited states of two dye molecules in
which excitation is transferred from a donor
molecule to an acceptor molecule without emission
of a photon. FRET is dependent on the inverse
sixth power of the intermolecular separation,
making it useful over distances comparable with
the dimensions of biological macromolecules. Thus,
FRET is an important technique for investigating
a variety of biological phenomena that produce
changes in molecular proximity.
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Energy transfer mechanism
Fluorescein
Rhodamine
Donor Acceptor
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Energy transfer mechanism
Fluorescein
hn
Rhodamine
Donor Acceptor
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Energy transfer mechanism
Fluorescein
Rhodamine
hn
Donor Acceptor
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Primary Conditions for FRET

• Donor and acceptor molecules must be in
• close proximity (typically 10100 Å).
• The absorption spectrum of the acceptor must
• overlap fluorescence emission spectrum of the
• donor.
• Donor and acceptor transition dipole
  orientations
• must be approximately parallel.

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Förster and Dexter mechanismsof energy transfer
The Dexter mechanism involves overlap of
wavefunctions and is essentially identical to the
transition moment for absorption with the
difference that the initial and final states
involve two molecules. DA DA The Forster
mechanism is a dipole-dipole mechanism that can
operate over long distances (up to 100 Å!).
This is the mechanism that is commonly used in
biology to determine the distance between two
molecules.
Stryer L, Haugland RP. PNAS 58, 719-726 (1967)
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The Förster Radius
The rate constant for energy transfer is kDA
(R0/R)6 The distance at which energy transfer is
50 is Ro 8.8 x 10-28k2t0-1n-4FJ(l)1/6
k - orientation factor (2/3 for an isotropic
sample) n - index of refraction F - quantum yield
of the donor Spectral overlap integral
is Donor Acceptor Ro (Å) Fluorescein
Tetramethylrhodamine 55 IAEDANS
Fluorescein 46
Wu P, Brand L. Anal Biochem 218, 1-13 (1994)
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Fluorescent resonant energy transfer probes of
lipid mixing
Membranes labeled with a combination of
fluorescence energy transfer donor and acceptor
lipid probes typically NBD-PE and N-Rh-PE,
respectively are mixed with unlabeled
membranes. Fluorescence resonance energy
transfer, detected as rhodamine emission at 585
nm resulting from NBD excitation at 470 nm,
decreases when the average spatial separation of
the probes is increased upon fusion of labeled
membranes with unlabeled membranes. The reverse
detection scheme, in which fluorescence resonance
energy transfer increases upon fusion of
membranes separately labeled with donor and
acceptor probes, has also proven to be a useful
lipid mixing assay.
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Energy transfer strategy for theobservation of
membrane fusion
ET is not observed because D and A are too far
apart
Energy transfer (ET) is observed because D and A
are close
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Example of a donor/acceptor pair
Nitrobenzoxadiazolyl (NBD) phosphatidyl
ethanolamine Donor
Rhodamine phosphatidyl ethanolamine
Acceptor
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Excimer probes of membrane fusion
Pyrene-labeled fatty acids can be
biosynthetically incorporated into viruses and
cells in sufficient quantities to produce the
degree of labeling required for long-wavelength
pyrene -excimer fluorescence. This excimer
fluorescence is diminished upon fusion of
labeled membranes with unlabeled membranes.
Fusion can be monitored by following the
increase in the ratio of monomer (400 nm) to
excimer (470 nm) emission, with excitation at
about 340 nm.
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Excimer emission
Excimer emission arises from the charge-transfer
emission of two molecules in an excited state
complex. 1) 2 mM pyrene (Argon) 2) 2 mM pyrene
(Air) 3) 0.5 mM pyrene (Argon) 4) 2 µM pyrene
(Argon)
Py Py hnblue Py Py PyPy - Py Py
hnred
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Excimer emission as a probe ofmembrane fusion
Excimer emission decreases after fusion