BCHE3030
Methods in
Biochemistry
Fluorescence
Technology
Wilson CY Lau
Assistant Professor
School of Life Sciences, CUHK
Science Center 184 Tel: 3943 6253
Email: [email protected]
BCHE3030 Methods in Biochemistry - Outline
Assignment – Fluorescence technology
In groups of 5-6, choose a recent (within the past 5 years) original research paper that utilized one of
the fluorescence techniques (see below) and write a summary and present a powerpoint presentation.
The summary should be between 750-1500 words and the presentation should be 10 mins max. Both
the summary and the powerpoint presentation should include the background, rationale and
methodology of the research study. The powerpoint slides should be submitted two days before the
date of the presentation.
Marking Scheme
Summary 4 %
Powerpoint slides 3 %
Oral presentation 3%
BCHE3030 Methods in Biochemistry - Outline
Assignment – Fluorescence technology
Please form your group by Jan 17 (next Tues)
Some Calcium signaling
Calcium as a secondary messenger
Calcium signaling has diverse fundamental roles in biology, ranging
from muscle contraction, metabolism, secretion, fertilization,
neuronal plasticity, cell division, phagocytosis, cell motility, and many
more
Spatial and temporal control are important
Hundreds of proteins have adapted to bind calcium over a million-fold
range of affinities (nM to mM)
Intracellular (100 nM free) vs extracellular (mM) Ca2+ concentrations
Organelles (i.e. mitochondria and ER) also have relatively high Ca2+
concentration (hundreds of micromolars)
Secondary messenger: signal
amplification
Some Calcium signaling
Some Origins of Ca2+ Imaging with Fluorescent Indicators
1980
Roger Y. Tsien EGTA has Ca2+/Mg2+ selectivity (Mg exits in an 10000:1 excess over Ca)
(1952-2016) Ca2+ has a larger ionic radius than Mg2+
Nobel Prize in The aliphatic amines critical for Ca binding are extremely basis (pKa of 9 and 8.6)
Chemistry 2008 Ca Kd of BAPTA (~100 nM) is similar to that of EGTA (~150 nM), Mg Kd of BAPTA is ~20
mM
Some Origins of Ca2+ Imaging with Fluorescent Indicators
BAPTA
Upon Ca2+ binding, absorbance of 254 nm => 203 nm
A few limitations
1) Proteins and nucleic acids absorb in the same region
2) Not fluorescent at visible wavelengths
3) Unable to cross cell membranes
Delivery of small molecules to
cells via lipophilic acetoxymethyl
(AM) esters
Another way is by microinjection,
which is undesirable
Some Fura-2: a calcium indicator
Some Origins of Ca2+ Imaging with Fluorescent Indicators
Ratio-Based Ca2+ Imaging
with the development of
Fura-2 (1985)
Binding of Ca induced
conformational rotation
of the aniline
Emission wavelength was
changed as a result of Ca
binding, but excitation
wavelength differs: Ca-
bound form (335 nm) or
the Ca-free form (362 nm)
Some Origins of Ca2+ Imaging with Fluorescent Indicators
Ratio-Based Ca2+ Imaging
with the development of
Fura-2 (1985)
By taking the ratio of the
emission intensity in cells
excited at the
wavelengths
corresponding to the Ca2+-
bound and -free forms of
fura-2, one could estimate
an actual
Ca2+ concentration in
living cells
Some Origins of Ca2+ Imaging with Fluorescent Indicators
Ratio-Based Ca2+ Imaging
with the development of
Fura-2
Advantages:
-Uneven loading of dye in
different cellular
compartments/tissues
-fluctuations in excitation
source intensity
-drifts in detector
sensitivity
-photobleaching
Some Origins of Ca2+ Imaging with Fluorescent Indicators
Some Origins of Ca2+ Imaging with Fluorescent Indicators
Structures of synthetic Ca indicators
Some Origins of Ca2+ Imaging with Fluorescent Indicators
Fluo-3
Intensity-based Ca Imaging
Unlike the case for fura-2, the Ca2+-induced change in the optical
properties of fluo- and rhod-type dyes is an increase in the
fluorescence quantum yield at a single wavelength, precluding
estimates of the actual Ca2+ concentration. However, fluo-2 and its
progeny had the advantage of matching commonly available
excitation and emission courses in microscopes worldwide, and the
ability to track changes in Ca2+ levels, in real time, using
commercially available microscopes was quickly adopted.
Some Spectrophotometer vs Spectrofluorometer
Spectrophotometer
A spectrophotometer is an analytical instrument
that can measure the concentration of a sample
by measuring light absorption
This instrument can operate at visible light, near UV,
and near IR lights, as well. We use a cuvette to
place the sample inside the instrument. Then a light
beam passes through the sample and diffracts into a
spectrum of wavelengths. Then the instrument
measures the intensities via a charge-coupled
device
Some Spectrophotometer vs Spectrofluorometer
Spectrofluorometer Obtain information about the concentration and
the chemical properties of the sample. In this
instrument, we need to select a certain excitation
wavelength. We can observe the emission at a
single wavelength, or we can just scan the
sample in order to record the intensity against the
wavelength. This is also called an emission
spectrum. We can use this instrument in
fluorescence spectroscopy.
Sensitivity of fluorescence detection is ~1000
times greater than absorption method;
fluorimetry has wider detection range and
higher specificity
Fluorescence Technology - Outline
Introduction to Fluorescence, Fluorescent Probes (Week 1)
Green Fluorescent Protein (Week 1)
Foster Resonance Energy Transfer, Real-time Polymerase Chain
Reaction (Week 2)
Flow Cytometry (Week 2)
Fluorescence Microscopy, Single-molecule Studies (Week 4)
Microscale Thermophoresis (Week 4)
Discussion on Fluorescence Techniques (Week 5, first 1/2)
Some Green Fluorescent Protein (GFP)
The bioluminescent Pacific jellyfish Aequorea
Victoria, source of the blue-luminescent
protein aequorin and its partner the Green
Fluorescent Protein
No well-accepted explanation why jellyfish glows
when it is alarmed (maybe to scare off
enemies?)
Aequorin releases blue light
Some Green Fluorescent Protein (GFP) upon binding with calcium.
The blue light is then
absorbed by the GFP, which
in turn gives off green light
GFP emits green light
without cofactors or
enzymes
A cofactor is a non-protein
chemical compound or metallic
ion that is required for an
enzyme's role as a catalyst (a
A fluorophore is a fluorescent chemical compound that catalyst is a substance that
can re-emit light upon excitations that occur due to a light increases the rate of a chemical
source. Chromophore is a part of a molecule that is reaction). Cofactors can be
responsible for the color of that molecule considered "helper molecules"
that assist in biochemical
transformations
Some The Nobel Prize in Chemistry 2008
Shimomura et al (1962)
discovered GFP as a
contaminant of aequorin
and proposes (1979) the
structure of the
chromophore
Prasher et al (1992) cloned
the GFP gene and Chalfie
(1994) proved GFP is able to
fluoresce without any
cofactors
Tslen (1994) showed that
the mechanism of GFP
chromophore is formed in a
chemical reaction which
requires oxygen, etc
Some Genetic tagging with GFP
Some GFP variants
Some Structural features of GFP
The pH sensitivity of all GFPs (excited at
488 nm) is attributed to the protonation of
the electron-rich, light-absorbing part of the
chromophore, which occurs at sub-
physiological pH
Some Structural features of GFP The crystal structure of GFP was solved in
1996. It has a unique soda can shape and
is made up of 11 beta-strands with an
alpha-helix running through the center
The chromophore is located in the
middle of the beta-barrel
An alpha helix containing the covalently
bonded chromophore 4-(p-
hydroxybenzylidene)imidazolidin-5-one
(HBI) running through the center
HBI: the modified form of the tripeptide
Ser65-Tyr66-Gly67
GFP catalyzes the formation of its own
chromophore
Some GFP maturation
Inward-facing sidechains of the barrel induce
specific cyclization reactions in Ser65–Tyr66–
Gly67 that induce ionization of HBI to the
phenolate form and chromophore formation. This
process of post-translational modification is
referred to as maturation
The hydrogen-bonding network and electron-
stacking interactions with these sidechains
influence the color, intensity and
photostability of GFP and its numerous
derivatives
The tightly packed nature of the barrel
excludes solvent molecules, protecting the
chromophore fluorescence from quenching
by water
Some Proposed biosynthesis of GFP chromophore
Some Chromophore environment
Besides the three residues that form the
chromophore, residues such as Gln94, Arg96,
His148, Thr203, and Glu222 all act as stabilizers.
The residues of Gln94, Arg96, and His148 are able
to stabilize by delocalizing the chromophore
charge
Arg96 is the most important stabilizing
residue due to the fact that it prompts the
necessary structural realignments that are
necessary from the HBI ring to occur
Major excitation peak at 395 nm and a
Some Some facts about GFP minor one at 475 nm
Emission peak at 509 nm
Internal chromophore without any
cofactors/substrates/enzymes
wtGFP has several drawbacks: dual
peaked excitation spectra, pH sensitivity,
chloride sensitivity, poor fluorescence
quantum yield, poor photostability, and
poor folding at 37C
Excites at 488 nm and emits at 509 nm
after S65T, also increased fluorescence
and photostability
F64L improves folding at 37, allowing it
to be used in mammalian cells
=> enhanced GFP (EGFP)
Some Ser65 mutation improves excitation spectra
Some Structural features of GFP
GFP and its variants are prone to forming
low-affinity dimers under physiological
conditions.
A206, L221, and F223 are important for
GFP head-to-tail dimerization
Introduction of a single mutation, A206K,
has been shown to disrupt hydrophobic
interactions in the region responsible for
GFP dimerization
Some GFP variants
Some GFP variants
GFP and its blue, cyan, and
yellow variants have found
widespread use as genetically
encoded indicators for tracking
gene expression and protein
localization and as
donor/acceptor pairs for FRET
T203Y mutation
Some Fluorescence properties of different GFP variants
Some Many tropical corals contain fluorescent proteins
Some The problems with DsRed
Tetramerization of DsRed
Slow maturation
Residual green fluorescent component
Some The palette of non-oligomerizing fluorescent proteins
tdTomato (tandem dimer)
Some Maturation time for fluorescent proteins
The final oxidation step is the rate-limiting step
The maturation time compromise our ability to accurately
monitor the dynamics of a variety of cellular processes
