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Transcript
Study of Protein Association by
Fluorescence-based Methods
Kristin Michalski
UWM RET Intern
In association with Professor Vali Raicu
Role of Proteins
• Proteins are complex molecules
that play many critical roles in
the body.
• They are required for the
structure, function, and
regulation of the body’s tissues
and organs.
• Proteins are made up of many of
smaller units called amino acids,
which are attached to one
another in long chains.
Role of Proteins
• Proteins are the
catalysts of
biological
functions.
• Also, proteins
never act alone.
For example, a growth hormone is a messenger protein made by the pituitary
gland. It regulates cell growth by binding to a protein called a growth hormone
receptor.
Why is it important to study protein
associations?
• Protein associations are known to occur,
but what mechanism causes proteins to
combine is not always understood.
• These associations occur at a distance of
~1 nm. Currently, there is no microscope
that can view these small interactions.
• How do we see these protein
associations?
Fluorescent Tagging
• Because these structures are so small, proteins
of interest are tagged with fluorescent markers in
order to study the proteins themselves and their
interactions in living cells based on the detection
of light from fluorescent tags.
What is fluorescence?
• Fluorescence is a process in which a molecule is excited by the
absorption of light. As the molecule moves to ground state, light is
emitted at lower energy (lower wavelength).
• This process is known as red-shifting.
Energy
lost
absorb
high
energy
photon
Emits lower
energy photon
Ground State
Fluorescence is a process by which
molecules absorb light of certain energy or
wavelength (e.g., blue), lose some energy
internally, and emit a less energetic photon
(e.g., green). This emission of light is
dependent on the chemical structure of the
molecule.
Process of tagging proteins
• Specific plasmids are chosen and inserted
into the cell to knock out the chromosome
and produce an altered chromosome.
Gene
STE 2
Ste
2P
Gene
responsible
for expression
of GFP
Process of tagging proteins
• The letters in the genome that express a specific amino acid on a
protein are found.
• This amino acid is cut out of the genome using enzymes and
replaced with the portion of the gene that expresses GFP (or YFP).
• The cells are then left to reproduce with this new coding.
Gene
Gene
STE 2
Ste
2P
Gene
responsible
for expression
of GFP
STE 2
Ste
2P
Fluorescent Emissions
• Because we can not view protein associations,
we must use a useful tool that can measure the
fluorescence of our tagged proteins.
• The tool that we use is Förster resonance
energy transfer or fluorescence resonance
energy transfer (FRET), a process in which an
excitation of a fluorescent molecule is
transferred non-radiatively to a nearby molecule.
How do we see fluorescence?
• There are two types of light
microscopy, Epifluorescence
and Confocal microscopy, used
to detect the light emitted by
fluorescent molecules tagged to
a selected cell structure of
interest.
• In this project, the
epifluorescence microscope was
used to locate the cells of
interest and the confocal was
used for the data collection.
Transmission Image
• Here is a transmission
image of the cells
expressing green
fluorescence
(fluorescence not seen
here).
• A transmission image
was recorded before
and after each data
collection to locate any
shifting of the cells.
How does a Confocal Microscope
work?
• The laser light reflects off of a dichroic mirror
and then hits two mirrors which are mounted on
motors; these mirrors scan the laser across the
sample.
• When laser light interacts with fluorophores in
the specimen, they emit light which is reflected
back through the beam path by the same mirrors
that are used to scan the excitation light from the
laser.
Confocal Microscope
• In a confocal laser scanning microscope a
laser is used to provide an excitation light
in order to get very high intensities that will
penetrate into specimens.
• One of the major benefits of using the
confocal microscope is it allows for optical
sectioning.
Confocal Microscope
Confocal Microscope
• The emitted light passes through the dichroic
mirror and is focused onto a pinhole.
• Because the pinhole diameter is small, very little
of the out of focus light emitted from the
fluorophores located above and below the focal
plane passes through the pinhole aperture.
• The light that passes through the pinhole is
measured by a detector, a photomultiplier tube.
Confocal Microscope
• The detector builds
up the image one
pixel at a time.
• This is collection of
proteins within the
cell. Because
proteins are
expected in the cell
wall, it makes sense
to see a brighter
image there.
Confocal Microscope
• In the
quantitative
analysis, the
area of each cell
is found. The
intensity within
that area is
measured,
determining the
total amount of
the fluorescence
in each cell.
FRET Studies
1. FRET can determine the energy-transfer
efficiency providing a measure the
intermolecular distances inside a protein
complex.
2. FRET can determine quantities of the
molecular interactions, the ratio of the
interacting protein complexes, and finally
the size of the oligomers (associated
proteins) formed.
FRET
• A donor protein (GFP) can “lose” energy if
an accepter is near.
• An accepter protein (YFP) can “gain”
energy if a donor is near.
GFP
YFP
FRET
• Some donor proteins (GFP) will associate with
acceptor proteins (YFP) and transfer energy
non-radiantively causing the intensity of the
donor fluorescence to be reduced and the
accepter’s fluorescence intensity to increase.
476 nm
476 nm
FRET
• Because the acceptor protein naturally emits
fluorescence, photobleaching is used to “bleach”
the acceptor.
• Once the YFP is bleached, FRET is used as a
tool to measure the amount of energy that is
emitted from GFP.
514 nm
YFP
YFP
FRET Quantitative Results
• Spectral
deconvolution of
composite
fluorescence
spectra of cells
expressing
Ste2p-GFP and
STE2p-YFP
FRET Quantitative Results
• The intensity of the FRET signal will be
dependent on the concentration of donor
and acceptors present.
• In FRET studies, fluorescence intensity of
samples is measured at several
wavelengths.
Studies of fluorescence on a
collection of cells
• The first part of the presentation explained
how fluorescence in measured on
individual cells but it would be useful to
study a collection of cells.
• This will be done using a duel Absorption
and Emission Spectrometer.
Spectrofluorometers using cell
suspended in liquid
• Using cells that are suspended in a liquid
medium, two things can be obtained.
• First, through the use of a absorption
spectrometer, the number of cells in the
suspension can be measured.
• Second, with the use of the emission
spectrometer, fluorescence of the cells can be
measured.
• Because of the dynamic nature of living cells,
absorption and emission of cell expression
should obtained within minutes of each other.
Spectrofluorometer
Mirror
Mirror
Absorption
PX-2
Xe
Lamp
Fluorescence
Cuvette
Holder
Beamsplitter
30R/70T
Lens
Filter
Spectrometer
USB200
OOIBase32 Software