green fluorescent protein a b/mb senior seminar brought to you by colm o’carroll
TRANSCRIPT
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
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 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