Transcript 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
• The use of GFP to monitor viral movement
in plants
The Green Fluorescent Protein
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
The Active Site
The Fluoropore Active Site
• Ser65-Tyr66-Gly67
• Deprotonated phenolate of Tyr66 is cause of
fluorescence
• Forster Cycle (1949-Theodor Forster)
• Proton transfer to His148
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