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

Useful GFP mutants

• Re-engineered GFP with preferred human codon usage

• 20 fold enhancement consistent with 20 fold increase of GFP protein levels

• GFP mutants can fluoresce different colors and be used simultaneously to monitor independent events in cells

• Some GFP mutants exhibit more rapid formation of fluorophore

Improved mutant GFPuv

• Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (black lines) GFPuv (purple lines). The emission data were obtained with excitation at 385 nm.

• Exhibiting lower toxicity in bacteria, GFPuv grows 2-3 times faster than wt GFP.

Advantages of GFP mutants in plants

• High levels of GFP do not interfere with transformation, regeneration, or growth

• Early nondestructive identification of transformed cells

• Developing and optimizing transformation methods

• Spatial and temporal gene expression at subcellular, cellular and plant levels

Studying virus invasion and spread in plant tissue

• Replaces marker protein -glucuronidase (GUS)

• Procedure safe for cells

• Requires only molecular oxygen for flourophore formation

Procedure

• Plants infected with PVX (Potato virus x-based vector)

• Containing various GFP inserts– PVX expressing free GFP gene– PVX expressing GFP PC (protein coat) fusion– PVX with PC deletion/GFP replacement – PVX with GFP fusion to movement proteins

Results indicated

• Free GFP expression-radial expansion

• GFP CP fusion cells possess a GFP ‘overcoat’

• PC Deletion/GFP replacement- fluorescence restricted to single inoculated cells

• GFP/MP fusion localized to plasmodesmata

Bibliography

• Nina, Haruki, et al. "Chemical nature of light emitter of Aequorea green fluorescent protein" (1996) Proceedings Natl. Acad. Sci. USA vol. 93 p.13671-13622

• Oparka, Karl, et al. "Using GFP to study virus invasion and spread in plant tissues"(1997) Nature vol. 388 p. 401-402

• Reid, Brian, Gregory Flynn. "Chromophore formation in Green Fluorescent Protein" (1997) Biochemistry vol. 36 p. 6786-6791

• Yang, F., L. Moss, G. Phillips. "The Molecular Structure of GFP" (1996) Nature Biotechnology vol. 14 p. 1219-1220

• Youvan, Douglas., Gregory Flynn. "Chromophore formation in Green Fluorescent Protein" (1997)

Biochemistry vol. 36 6786-6791

Sources on the World Wide Web

• Medical College of Wisconsin (www.biochem.mcw.edu/science_ed/Pages/gfp/index.html

• Clonetech (www.gfp.clontech.com)

• www.biorad.com/889168.html

• www.bio.cmu.edu/Courses/03740/GFPTest/GFP.html