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, and 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
ex) Single point mutation (S65T) reported in Nature (1995)
- This mutation dramatically improved the spectral characteristics of GFP, resulting in
increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm,
with the peak emission kept at 509 nm.
 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.
Fluorescent proteins
• Revolutionized medical and biological sciences by providing a way to
monitor how individual genes are regulated and expressed within a living
cell ; Localization and tracing of a target protein in the cells
• 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)
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)
Chromophore formation in GFP
GFP and chromophore
-
Covalently bonded chromophore : 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI).
HBI is nonfluorescent in the absence of the properly folded GFP scaffold and exists mainly in the
unionized phenol form in wt-GFP.
- Maturation (post-translational modification) : Inward-facing side chains of the barrel induce
specific cyclization reactions in the tripeptide Ser65–Tyr66–Gly67 that induce ionization of HBI to
the phenolate form and chromophore formation.
- The hydrogen-bonding network and electron-stacking interactions with these side chains influence
the color, intensity and photo-stability of GFP and its numerous derivatives
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.