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)

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 Sci-ence.

• Although this wt GFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, poor photo-stability and poor folding at 37°C.

• 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, diagnos-

tics, genetics, etc. • 2008 : Martin Chalfie, Osamu Shimomura and Roger Y. Tsien shared the

Nobel Prize in Chemistry for their discovery and development of the fluo-rescent proteins.

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)

GFP and fluorophore

wtGFP : Ser(65)-Tyr(66)-Gly(67)

Diverse Fluorescent Proteins by Protein Engineering

The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent pro-teins.

Fluorescence emission by diverse fluorescent Proteins

a) Normalized absorption and b) fluorescence profiles of rep-resentative fluorescent pro-teins:

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 Clon-tech.

Absorption and emission spectra