Presentation on theme: "Fluorescent proteins Green Fluorescence Protein (GFP) from jellyfish : Revolutionized medical and biological science by providing a way to monitor how."— Presentation transcript:
1 Fluorescent proteinsGreen Fluorescence Protein (GFP) from jellyfish : Revolutionized medical and biological science by providing a way to monitor how individual genes are regulated and expressed within a living cell ; Localization and tracing of a target proteinWidespread use by their expression in other organisms as a reporterUsually fused to N- or C-terminus of proteins by gene manipulationKey internal residues are modified during maturation to formthe p-hydroxybenzylideneimidazolinon chromophore, located in the central helix and surrounded by 11 ß-strands (ß-can structure)GFP variants : BFP, CFP, YFPRed fluorescent protein from coral reef : tetrameric, slow maturation- Monomeric RFP by protein engineeringQuantum 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.Utility as a tool for molecular biologists was not realized until 1992 whenDouglas 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 toseveral 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 : published in Science.Although this wt-GFP was fluorescent, it had several drawbacks:dual peaked excitation spectra, poor photo-stability, and poor folding at 37°C.
3 1996 : Crystal structure of a GFP Providing a vital background on chromophore formation and neighboringresidue interactions. Researchers have modified these residues usingprotein 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 victoriaA tightly packed -can (11 -sheets) enclosing an -helix containing the chromophore238 amino acidsChromophoreCyclic 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)
6 GFP and chromophoreCovalently 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 theunionized phenol form in wt-GFP.Maturation (post-translational modification) : Inward-facing side chains of the barrel inducespecific cyclization reactions in the tripeptide Ser65–Tyr66–Gly67 that induce ionization of HBI tothe phenolate form and chromophore formation.The hydrogen-bonding network and electron-stacking interactions with these side chains influencethe color, intensity and photo-stability of GFP and its numerous derivatives
7 Diverse Fluorescent Proteins by Protein Engineering wtGFP : Ser(65)-Tyr(66)-Gly(67)
8 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.
10 Absorption and emission spectra a) Normalized absorption andb) 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.