Fluorescent proteins

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
- The funding for this project had run out, so Prasher sent cDNA samples to several
• 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
• 238 amino acids
• Chromophore
– Cyclic tripeptide derived from
• 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
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