This document discusses fluorescent proteins, which are used to track and visualize proteins in living cells. It describes the desirable characteristics of fluorescent proteins and reasons for developing new variants, such as brighter fluorescence, different spectra, and greater photostability. Examples of new fluorescent proteins discussed include variants of GFP, CFP, YFP, and RFP with improved properties for multi-color imaging and FRET experiments. The goal is to generate fluorescent markers that better label proteins and reveal biological events within cells.
2. Aims
Applications with fluorescent proteins
Properties of fluorescent proteins
Reasons for upgrading fluorescent proteins
Examples of new fluorescent proteins
3. Why do we use fluorescent
proteins?
To track and quantify proteins
To watch protein-protein interactions
To describe biological events and signals in a
cell
4. Characteristics of
Fluorescent Proteins
Expressed efficiently
No phototoxicity
Bright enough
Sufficient photostability
No oligomerization
Minimal overlap in excitation and
emission profile
5. Reasons for New FPs
For brighter fluorescence
improving quantum yield
higher extinction coefficient
quicker maturation
6. Reasons for New FPs
To change absorbance and emission
spectra
less spectral overlap
better spectral separation
Longer fluorescence lifetime
Less photobleach
7. Reasons for New FPs
Less sensitive to environment
pH resistance
ions
Deeper tissue penetration
8. New Fluorescent Proteins
The discovery of GFP from jellyfish
Mutagenesis studies on GFP
New fluorescent proteins
9. Blue Fluorescent Protein
(BFP)
Shifts in absorbance and emission spectra
First used in multicolour imaging and FRET
BUT,
Dim
Photobleach easily
13. Yellow Fluorescent Protein
(YFP)
Citrine and Venus
Chloride sensitivity eliminated
Sensitivity to pH changed
Photobleaching improved
14. Red Fluorescent Proteins
(RFP)
From other marine organisms
Discosoma DsRed
Heteractis crispa HcRed
Most suitable red markers
15. Red Fluorescent Proteins
(RFP)
DsRed
needs incubation at 37ºC
obligate tetramer
Minimizing oligomerization
red fluorescent tandem dimers
Mrfp1 completely monomeric
matures quickly
25 nm longer wavelengths
16. New Fluorescent Proteins
New approach for monomeric red
fluorescent proteins
Replacing N terminus of mRFP1 with GFP
Adding C terminus of GFP to mRFP1
17. New Fluorescent Proteins
All variants are brighter than mRFP1
(except mHoneydew, mBanana,
mTangerine)
mOrange is the brightest but sensitive to
pH.
mCherry is the most photostable
18.
19.
20. New Fluorescent Proteins
Protein lifetimes and turnover rates
First little initial fluorescence with
excitation wavelength
Then high fluorescence with different
wavelength
PA-GFP, Kaede, KFP1
21.
22. New Fluorescent Proteins
PA-GFP developed from GFP
increase in fluorescence
when illuminated at 413 nm
Kaede identified from T. geoffroyi
converted from a green to a
red fluorescent protein by irridation with
350-400 nm
KFP1 from Anemonia sulcata
23. Summary
We use the fluorescence proteins to see
changes in cells
Fluorescence proteins have common
properties
Improving fluorescence proteins for
better imaging
Examples of new fluorescent proteins
24. References
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