Green

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Green

  1. 1. Green Fluorescent Protein a B/MB senior seminar brought to you by Colm O’Carroll
  2. 4. This presentation will cover <ul><li>The structural aspects of GFP which make fluorescence possible </li></ul><ul><li>The advantages of using GFP and GFP mutants over other fluorescent markers </li></ul><ul><li>The use of GFP to monitor viral movement in plants </li></ul>
  3. 5. The Green Fluorescent Protein
  4. 6. GFP’s unique structure <ul><li>Composed of 238 amino acids </li></ul><ul><li>“ Paint in a can” </li></ul><ul><li>Each monomer composed of a central  -helix surrounded by an eleven stranded cylinder of anti-parallel  -sheets </li></ul><ul><li>Cylinder has a diameter of about 30A and is about 40A long </li></ul><ul><li>Fluorophore located on central helix </li></ul>
  5. 7. The Active Site
  6. 9. The Fluoropore Active Site <ul><li>Ser65-Tyr66-Gly67 </li></ul><ul><li>Deprotonated phenolate of Tyr66 is cause of fluorescence </li></ul><ul><li>Forster Cycle (1949-Theodor Forster) </li></ul><ul><li>Proton transfer to His148 </li></ul>
  7. 10. Fluorophore formation <ul><li>One limitation of wtGFP is its slow rate of fluorescence acquisition in vivo </li></ul><ul><li>Renaturation most likely by a parallel pathway </li></ul><ul><li>Oxidation of Fluoropore (2-4 hours) </li></ul><ul><li>Two step process </li></ul>

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