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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

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Green Fluorescent Protein

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  1. Green Fluorescent Protein a B/MB senior seminar brought to you by Colm O’Carroll

  2. 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

  3. The Green Fluorescent Protein

  4. 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

  5. The Active Site

  6. The Fluoropore Active Site • Ser65-Tyr66-Gly67 • Deprotonated phenolate of Tyr66 is cause of fluorescence • Forster Cycle (1949-Theodor Forster) • Proton transfer to His148

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Studying virus invasion and spread in plant tissue • Replaces marker protein -glucuronidase (GUS) • Procedure safe for cells • Requires only molecular oxygen for flourophore formation

  12. 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

  13. 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

  14. 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

  15. 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

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