BIOL 5060 Lecture Notes - Lecture 19: Escherichia Coli, Lac Repressor, Model Organism
7 pages12 viewsSpring 2017
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4/4_RecombinantDNATechnology_Hoffman4/4/2017 3:00:00 PM
Fluorescent Molecules (GFP)
• Will lose their fluorescence if a light is shined on them too much
• Also takes a moment for you to be able to see the difference—for
the fluorescence to show
Where in a cell is “my” protein
• Fusion to GFP (Verify if protein is still functional)
• Live cell microscopy
Do two proteins co-localize?
• Are they in the same location in the cell
• GFP to one protein
• YFP to the second protein
• Filters to see the two different emission wavelengths
• Seeing them in the same location doesn’t really mean that much
though, what we really want to know is if they work together.
• …
Q: Do my proteins interact or when do they interact? A: FRET
• We can add compounds/ molecules/ etc. to interact… see if it
changes the function
• FRET= fluorescent resonance energy transfer
• Can detect when CFP and YFP are within 10 nm of each other
o 10 nm= diameter of a typical protein
o this gets us down to the level of protein-protein interactions
• Protein X- CFP and Protein Y- YFP
Excitation
Emission
GFP
488
507
CFP
434
476
YFP
514
527
• All very different wavelength
• For CFP- we give it photons coming in at 434 and we should read
emission being emitted at 476
• YFP emission at 527
o The two emissions are too far from each other now in
distance
• But maybe they interact with each other in different conditions
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o There is some kind of energy transfer from one protein to the
other
• If CFP is excited at 434 and YFP is receiving energy, then CFP is
losing energy we would see a reduction in the CFP (476) Emission
and an increase in the YFP (527) emission
o This would show that these two proteins are within the typical
distance of a protein-protein interaction
o **must excite from the lower wavelength to the higher
FRET!: is a mechanism describing energy transfer between two
lightsensitive molecules (chromophores).[1] A donor chromophore, initially in
its electronic excited state, may transfer energy to an acceptor chromophore
through nonradiative dipole–dipole coupling.[2] The efficiency of this energy
transfer is inversely proportional to the sixth power of the distance between
donor and acceptor, making FRET extremely sensitive to small changes in
distance.[3]
• Measurements of FRET efficiency can be used to determine if
two fluorophores are within a certain distance of each other.[4]
Another use of FRET- as a sensor = FRET Sensors
• Detects changes in protein-protein interaction or a change in the
conformation of a protein
• FRET based proteins that are known to interact or lose an
interaction based on something else going on in the cell (gives a
measurement of what is disturbing the interaction in the cell)
• Example: Cyclic AMP detection (can be used for calcium detection,
any of a number of small molecules that would change a protein-
protein interaction or the conformation of a protein)
o PKA forms when there are low cAMP levels
o When there are high cAMP levels- the regulatory units binds
cAMP and changes its shape
o This protein-protein interaction is controlled by cAMP levels
o One way of detecting cAMP levels is by putting CFP on the
regulatory unit and YFP on the catalytic unit
o Under low cAMP levels you would see a FRET signal; under
high cAMP levels you would see a loss of FRET signal
3 more uses of fluorescence in molecular cell biology
FRAP- fluorescence recovers alto photo-bleaching
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