Fluorescent proteins and their applications

1. Fluorescent proteins and their applications • Fluorescence principles • The Green Fluorescent Protein • The DsRed Fluorescent Protein • Applications of fluorescent proteins

2. Excitation and emission spectra of fluorophores Molecular or atomic orbital energy levels Non-radiative transition Eexcitation = hc/excitation Eemission = hc/émission Non-radiative transition Resting state Excited state • emission spectrum: lexcitation fixed; lemission variable • excitation spectrum: lemission fixed; lexcitation variable

3. Jablonski diagram 2 E2 1 : absorption 2 : non-radiative transition 3 : emission 1 3 E1 2 • Absorption spectrum • Fluorescence spectra Fluorescence intensity O.D. absorption emission excitation absorption excitation emission wavelength wavelength Stockes shift

4. Aequoreavictoria Luminescence of aequorin and GFP Discovery of Aequorin and the Green Fluorescent Protein (GFP) PDB structure : 1EMA Aequoria victoria GFP

5. The GFP fluorophore cyclization oxidation pH sensitivity Niwa, Haruki et al. (1996) Proc. Natl. Acad. Sci. USA 93, 13617-13622

6. Discovery of Red Fluorescent Proteins PDB structure : G7K Discosoma sp DsRed protein Discosoma reef coral http://www.reefpedia.com/index.php/Discosoma • Red fluorescence • Long maturation time • Tetrameric protein •  Directed mutations : set of yellow to far-red fluorescent proteins

7. DsRed structure Auto-oxidative fluorophore generation green fluorescence DsRed chromophore : QYG blocked in K83R mutant Comparison of GFP and DsRed polypeptide structure red fluorescence Yarbrough & al. 2001 Proc. Nat. Ac. Sci.98 : 462-67 Ser65 GFP chromophore : SYG

8. Visualization of proteins of interest, in fusion with fluorescent proteins Protein of interest codon 2 codon 3 GFP codon 3 codon 2 ATG stop promoter 383 445 439 476 548 562 587 610 590 649 Nathan Shaner et al (2004) Nature Biotech. 22: 1567-1572 Lei Wang et al (2004) Proc. Natl. Acad. Sci. USA 101: 16745-16749 From R. Tsien NP lecture

9. Reporter genes A DNA of interest can be integrated in the genome of cells or organisms Protein of interest GFP Localization of a protein of interest Promoter of interest Place and time where a promoter is active Transcription factor activity Labeling of specific cells in an organism GFP Polypeptide whose conformation is sensitive to a molecule of interest In vivo measure of the concentration of a molecule of interest (FRET, FLIM) Promoter CFP YFP Polypeptide able to bind the phosphorylated form of the substrate In vivo measure of the activity of a kinase of interest (FRET, FLIM) Kinase substrate Promoter CFP YFP

10. Osamu Shimomura NP 2008 for the discovery of GFP and aequorin Martin Chalfie NP 2008 for the first use of GFP as a reporter in the nematode Robert Tsien NP 2008 for the development of intracellular fluorescent indicators, especially for Ca2+ Douglas C. Prasher Cloned in 1992 the GFP gene (Gene, 111 (1992) 229-233 ) http://www.telegraph.co.uk/news/worldnews/northamerica/usa/3178845/The-scientist-the-jellyfish-protein-and-the-Nobel-prize-that-got-away.html#