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Green Fluorescent Protein

Green Fluorescent Protein. a B/MB senior seminar brought to you by Colm O’Carroll. This presentation will cover . The structural aspects of GFP which make fluorescence possible The advantages of using GFP and GFP mutants over other fluorescent markers

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Green Fluorescent Protein

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  1. Green Fluorescent Protein a B/MB senior seminar brought to you by Colm O’Carroll

  2. This 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 plants

  3. The Green Fluorescent Protein

  4. GFP’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 helix

  5. The Active Site

  6. The Fluoropore Active Site • Ser65-Tyr66-Gly67 • Deprotonated phenolate of Tyr66 is cause of fluorescence • Forster Cycle (1949-Theodor Forster) • Proton transfer to His148

  7. Fluorophore 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 process

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