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Lecture 19

BIOL 5060 Lecture Notes - Lecture 19: Escherichia Coli, Lac Repressor, Model Organism

7 pages21 viewsSpring 2017

Course Code
BIOL 5060

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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 differencefor
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
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
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
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 dipoledipole 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
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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