New Fluorescent Proteins S. Semih Ekimler(3)

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