Green Fluorescent ProteinSlide 2Slide 3This presentation will coverThe Green Fluorescent ProteinGFP’s unique structureThe Active SiteSlide 8The Fluoropore Active SiteFluorophore formationSlide 11Slide 12Slide 13Green Fluorescent Proteina B/MB senior seminarbrought to you by Colm O’CarrollThis presentation will cover •The structural aspects of GFP which make fluorescence possible•The advantages of using GFP and GFP mutants over other fluorescent markers•The use of GFP to monitor viral movement in plantsThe Green Fluorescent ProteinGFP’s unique structure •Composed of 238 amino acids•“Paint in a can”•Each monomer composed of a central -helix surrounded by an eleven stranded cylinder of anti-parallel -sheets•Cylinder has a diameter of about 30A and is about 40A long•Fluorophore located on central helixThe Active SiteThe Fluoropore Active Site•Ser65-Tyr66-Gly67 •Deprotonated phenolate of Tyr66 is cause of fluorescence •Forster Cycle (1949-Theodor Forster)•Proton transfer to His148Fluorophore formation•One limitation of wtGFP is its slow rate of fluorescence acquisition in vivo•Renaturation most likely by a parallel pathway•Oxidation of Fluoropore (2-4 hours)•Two step
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