Upload
semih-ekimler
View
767
Download
3
Tags:
Embed Size (px)
Citation preview
NEW FLUORESCENT PROTEINS
S. Semih EKIMLER
Aims
Applications with fluorescent proteins
Properties of fluorescent proteins
Reasons for upgrading fluorescent proteins
Examples of new fluorescent proteins
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
Characteristics of Fluorescent Proteins
Expressed efficiently No phototoxicity Bright enough Sufficient photostability No oligomerization Minimal overlap in excitation and emission
profile
Reasons for New FPs
For brighter fluorescence
improving quantum yield
higher extinction coefficient
quicker maturation
Reasons for New FPs
To change absorbance and emission spectra
less spectral overlap
better spectral separation
Longer fluorescence lifetime
Less photobleach
Reasons for New FPs
Less sensitive to environment
pH resistance
ions
Deeper tissue penetration
New Fluorescent Proteins
The discovery of GFP from jellyfish
Mutagenesis studies on GFP
New fluorescent proteins
Blue Fluorescent Protein (BFP)
Shifts in absorbance and emission spectra First used in multicolour imaging and FRET
BUT,
Dim Photobleach easily
Cyan Fluorescent Protein (CFP)
Has a spectra between BFP and eGFP Brighter Displays more photostability
Cyan Fluorescent Protein (CFP)
A new version of CFP Cerulean Brighter Improves the signal/noise of FRET
Yellow Fluorescent Protein (YFP)
The absorption amd emission spectra are shifted to red wavelengths
Imaging partner of CFP (FRET)
Yellow Fluorescent Protein (YFP)
Citrine and Venus
Chloride sensitivity eliminated
Sensitivity to pH changed
Photobleaching improved
Red Fluorescent Proteins (RFP)
From other marine organisms Discosoma DsRed
Heteractis crispa HcRed Most suitable red markers
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
New Fluorescent Proteins
New approach for monomeric red fluorescent proteins
Replacing N terminus of mRFP1 with GFP Adding C terminus of GFP to mRFP1
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
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
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
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
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