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