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.

1. S. Semih EKIMLER

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

10. Cyan Fluorescent Protein
(CFP)
 Has a spectra between BFP and eGFP
 Brighter
 Displays more photostability

11. Cyan Fluorescent Protein
(CFP)
 A new version of CFP Cerulean
 Brighter
 Improves the signal/noise of FRET

12. Yellow Fluorescent Protein
(YFP)
 The absorption amd emission spectra
are shifted to red wavelengths
 Imaging partner of CFP (FRET)

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

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

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
 Lippincott-Schwartz, J., et al., 2003. Development adn Use of Fluorescent Protein
Markers in Living Cells. Science, 300(87), p.87-91
 Miyawaki, A., Sawano, A., Kogure, T., 2003. Lightening up cells: labelling proteins with
fluorophores. Nature Cell Biology, 5, p.S1-S7
 Patterson, G.H., 2004. A new harvest of fluorsecent proteins. Nature Biotechnology,
22(12), p.1524-1525
 Rizzo, M.A., Springer, G.H., Granada, B., Piston, D.W., 2004. An improved cyan
fluorescent protein variant useful for FRET. Nature Biotechnology, 22(4), p.445-449
 Sekar, R.B., Periasamy, A., 2003. Fluorescence resonance energy transefer (FRET)
microscopy imaging of live cell protein locations. The Journal of Cell Biology, 160(5),
p.629-633
 Shaner, N.C., Steinbach, P.A., Tsien, R.Y., 2005. A guide to choosing fluorescent
proteins. Nature Biotechnology, 2(12), p.905-909
 Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N.G., Palmer, A.C.,
Tsien, R.Y., 2004. Improved monomeric red, orange and yellow fluorescent proteins
derived from Discosoma sp. red fluorescent proteins. Nature Biotechnology, 22(12),
p.1567-1572
 Zhang, J., Campbell, R.E., Ting, A.Y., Tsien, R.Y., 2002. Creating New Fluorescent
Probes for Cell Biology. Nature, 3, p.906-918