1. Fluorescent proteins Green Fluorescence Protein (GFP) from jellyfish : Revolutionized medical and biological science by providng a way to monitor how individual genes are regulated and expressed within a living cell ; Localization and tracing of a target protein Widespread use by their expression in other organisms as a reporter Usually fused to N- or C terminus of proteins by gene manipulation Key internal residues are modified during maturation to form the p-hydroxybenzylideneimidazolinon chromophore, located in the central helix and surrounded by 11 ß-strands (ß-can structure) GFP variants : BFP, CFP, YFP Red fluorescent protein from coral reef : tetrameric, slow maturation - Monomeric RFP by protein engineering Quantum yield : 0.17 (BFP) ~ 0.79 (GFP)

2. History of Fluorescent Proteins 1960s : Curiosity about what made the jellyfish Aequorea victoria glow  Green protein was purified from jellyfish by Osamu Shimomura in Japan. Its utility as a tool for molecular biologists was not realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wt GFP in Gene. - The funding for this project had run out, so Prasher sent cDNA samples to several labs. 1994 : Expression of the coding sequence of fluorescent GFP in heterologous cells of E. Coli and C. elegans by the lab of Martin Chalfie : publication in Science. Although this wt GFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, poor photo-stability and poor folding at 37°C.

3. 1996 : Crystal structure of a GFP  Providing vital background on chromophore formation and neighboring residue interactions. Researchers have modified these residues using protein engineering (site directed and random mutagenesis)  Generation of a wide variety of GFP derivatives emitting different colors ; CFP, YFP, CFP by Roger Y. Tsien group  Applications in many areas including cell biology, drug discovery, diagnostics, genetics, etc. 2008 : Martin Chalfie, Osamu Shimomura and Roger Y. Tsien shared the Nobel Prize in Chemistry for their discovery and development of the fluorescent proteins.

4. GFP (Green Fluorescent Protein) Jellyfish Aequorea victoria A tightly packed  -can (11  -sheets) enclosing an  -helix containing the chromophore 238 amino acids Chromophore – Cyclic tripeptide derived from Ser(65)-Tyr(66)-Gly(67) Wt GFP absorbs UV and blue light (395nm and 470nm) and emits green light (maximally at 509nm )

5. GFP and fluorophore

6. wtGFP : Ser(65)-Tyr(66)-Gly(67) Diverse Fluorescent Proteins by Protein Engineering

7. The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins. Fluorescence emission by diverse fluorescent Proteins

9. a ) Normalized absorption and b) fluorescence profiles of representative fluorescent proteins: cyan fluorescent protein (cyan), GFP, Zs Green, yellow fluorescent protein (YFP), and three variants of red fluorescent protein (DS Red2, AS Red2, HC Red). From Clontech. Absorption and emission spectra