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:
1Fluorescent 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)
2History 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.
31996 : 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.
4GFP (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)
6GFP 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
7Diverse Fluorescent Proteins by Protein Engineering wtGFP : Ser(65)-Tyr(66)-Gly(67)
8Fluorescence 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.
10Absorption 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.