Download 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
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 xbased vector)
• Containing various GFP inserts
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–
–
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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