Green Fluorescent Protein

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Green Fluorescent Protein

• a B/MB senior seminar
• brought to you by Colm OCarroll

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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

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The Green Fluorescent Protein
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GFPs 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

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The Active Site
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The Fluoropore Active Site

• Ser65-Tyr66-Gly67
• Deprotonated phenolate of Tyr66 is cause of
  fluorescence
• Forster Cycle (1949-Theodor Forster)
• Proton transfer to His148

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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

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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

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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.

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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

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Studying virus invasion and spread in plant tissue

• Replaces marker protein ?-glucuronidase (GUS)
• Procedure safe for cells
• Requires only molecular oxygen for flourophore
  formation

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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

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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

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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

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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