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