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 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.
• 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.
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
Diverse Fluorescent Proteins by Protein Engineering
wtGFP : Ser(65)-Tyr(66)-Gly(67)
Fluorescence emission by diverse fluorescent Proteins
The diversity of genetic mutations is illustrated by this San Diego beach scene
drawn with living bacteria expressing 8 different colors of fluorescent proteins.
Absorption and emission spectra
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.