Green Fluorescent Protein

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Green Fluorescent Protein

• a B/MB senior seminar
• brought to you by Colm OCarroll

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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

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The Green Fluorescent Protein
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GFPs 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

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The Active Site
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The Fluoropore Active Site

• Ser65-Tyr66-Gly67
• Deprotonated phenolate of Tyr66 is cause of
  fluorescence
• Forster Cycle (1949-Theodor Forster)
• Proton transfer to His148

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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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