Review seminar gfp
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Transcript of Review seminar gfp
Presented by: Bhagyashree pareekM.Sc. IV Semester
Presented to: Dr. Ameeta sharma
THE IIS UNIVERSITY
Green fluorescent protein (GFP) is a protein composed of 238 amino acid residues
Exhibits bright green fluorescence
GFP was first isolated from the jellyfish Aequorea victoria GFP and GFP like proteins also found in
-Star coral -Strawberry anemone -Flat lettuce coral
INTRODUCTION
Most studied and exploited protien
Marker of the twenty-first century
Molecule of the twenty first century
Microscope of this century
NATURAL FUNCTION
Electron Donar
Burglar Alarm Hypothesis
MECHANISM OF GLOW Jellyfish produces green bioluminescence from small
photoorgans located on its umbrella Two protiens
-Aequorin &
-Green Fluorescent Protein
Aequorea releases calcium ions --bioluminesce.
Bind to a protein Aequorin, which release blue light upon calcium binding.
The blue light is absorbed by Green Fluorescent Protein, giving off the green light
Discovery and Major Milestone Osamu Shimomura
Shimomura intersted in the bioluminescence of the crystal jellyfish, Aequorea victoria.
First person to isolate GFP and to find out which part of GFP was responsible for its fluorescence
Douglas Prasher GFP as a tracer molecule.
GFP to report when a protein was being made in a cell
1990, Cloned Gene 0f GFP
1992 he published paper in the Gene, reported the cloning of GFP and the sequence of the 238 amino acids in GFP
Martin Chalfie
GFP as gene expression signal in Caenorhabitis elegans
Incorporated the GFP gene into E.coli
ROGER TSEIN
Engineered different mutants of GFPwith new optical properties {increased fluorescence, photostability and a shift of the major excitation peak}
Described the structural detail of GFP.
Explanation of mechanisms of GFP fluorescence
Osamu Shimomura discovered green fluorescent protein (GFP) in the small glowing jellyfish Aequorea victoria
Martin Chalfie introduced using of green fluorescent protein as a marker for gene expression
Roger Y. Tsien engineered different mutants of GFP with new optical properties (increased fluorescence, photostability and a shift of the major excitation peak ) and contributed to the explanation of mechanismus of GFP fluorescence
Nobel Prize in Chemistry in 2008
Structure
238 aminoacids
Beta pleated sheets and alpha helices
Crystal structure of GFP by Orm¨o et al and by Yang et al
Multiple Anomalous Dispersion of Selenomethionine groups
Crystal Structure
Beta-can shape.
11 beta-strands make up the beta-barrel and an alpha-helix runs through the center.
Chromophore is in the middle of the beta-barrel, it is occasionally referred to as the “light in the can”
STRUCTURE
Representation of the GFP beta-barrel. Beta sheets are GREEN, helices RED and connecting loops BLUE
TERTIARY STRUCTURE
The Fluorophore--Center of GFP
Ser-Tyr-Gly sequence positioned 65-67
Fluorophore “p-hydroxybenzylidene-imidazolidone”
Fluorophore is generated by auto-catalytic process
No co-factors or enzymatic components required
Biosynthesis of the Fluorophore
Rapid cyclization between Ser65 and Gly67 form an imidazolin-5-one intermediate
Dehydration of TYR-66
Oxygenation of the Tyr66 side chain by O2
Gly67 very important formation of the fluorophore
The reaction is thermosensitive
GFP is quite thermostable
Mechanism for Intramolecular biosynthesis of the GFP
chromophore
Fluorescent Spectrum of GFP• Two excitation peaks
– Major one at 395 nm
– Minor one at 475 nm
• Extinction coefficient of 30,000 and 7,000 M-1 cm-1
• Emission peak is at 509 nm
• GFP from the sea pansy exhibits a single major excitation peak at 498 nm
Fluorescence excitation (full-line curve) and emission (dashed curve) spectra of native GFP from Aequorea victoria (Tsien et al., 1998).
APPLICATIONS OF GFP 1.Fusion Tag
2.Reporte Gene
3. Fluorescence Resonance Energy Transfer
4. Photobleaching
5. Brainbow
6. Cancer Research
7. GFP Pets
8. HIV Virulence
9. GFP Ice-cream
GFP in Fusion Tag
Most successful and numerous class of GFP applications
GFP Gene fused to gene for endogenous protein
Chimera expressed in the cell or organism.
Fusion protein with normal functions and localizations of the host protein but is now fluorescent.
Tagged to every major organelle of the cell
GFP as Fusion Tag
Most successful and numerous class of GFP applications
GFP Gene fused to gene for endogenous protein
Chimera expressed in the cell or organism.
Fusion protein with normal functions and localizations of the host protein but is now fluorescent.
Tagged to every major organelle of the cell
Why GFP as Tracer molecule
• Small protein (MW 26.9 K Da)
• Post translational maturation
• Gene cloned
• No enzyme or cofactor required to produce flouescence
• Non-toxic
GFP as Reporter GeneSucessfully report the gene expression
Qualitatively and quantitatively
GFP has been used to detect gene expression in vivo especially in the nematode •Caenorhabditis elegans
GFP AS photbleaching
Photobleaching can be used to investigate protein dynamics in living cells.
GFP as FRET
Fluorescence Resonanc Energy Transfer
It is Non-radioactive exchange of Energy from excited donar to an acceptor fluorephore
Range is 10-100 Å
very useful study for protein-protein interaction
Study the protien interaction
BRAINBOW
• Mapping of individual neurons of a brain with fluorescent proteins
• Information about brain's intricate connections
• Varying the amount of red, green, and blue derivatives of green fluorescent protein
• Glow with specific colors under a light source
• Jeff W. Lichtman and Joshua R. Sanes
BRAINBOW
Cancer Research In vivo optical imaging of cancer
Study the metastatic movements of the cancerous cells
GFP Pets
HIV Virulence
GFP Ice-cream
Non-fluorescent and reversibly photoswitchable
proteinsFirst example: Pocilloporin
Absorbs Green or Yellow Light and appears Purple or Blue
In these proteins resting stage is brightly fluorescent.
Conclusion• GFP is a wonderful molecule
• Discovered from the depths of the oceans and brought revolutionary light in molecular biology
• Gfp can be used as a flourescent tag to any protien of intrest to study its synthesis and the intracellular traficking.
• Providing new insights in understanding the internal workings of the cell
Bibliography• http://micro.magnet.fsu.edu/primer/techniques/
fluorescence/fluorescentproteins/fluorescentproteinshome.html
• http://www.bioc.rice.edu/Bioch/Phillips/Papers/gfpbio.html• http://www.cbc.ca/news/ • http://www.coloumbia.edu • http://www.conncoll.edu/ccacad/zimmer/GFP-ww• http://www.gonda.ucla.edu/bri_core/gfp.htm• http://www.jic.ac.uk/microscopy/more/T5_9.htm• http://www.pnas.org • http://www.sciencedaily.com/articles/b/
bioluminescence.htm>• http://zeiss-campus.magnet.fsu.edu
Continued.....• Ando R., Baird, G.S. (2002) Fluorescence Microscopy Proc. Natl. Acad. Sci. USA 99:
12651-12656• Baird, G. S., Zacharias, D. A., Tsien, R. Y. Green Fluorescent Protein. Proc. Natl.
Acad. Sci. U.S.A., 97:11984-11989.• Baird, G.S. et al. (2000) Use Of Green Fluorescent protein Proc. Natl. Acad. Sci. 97:
11984-11989.• Beavis, A.; Kalejta, R.(1999). Applicatons of Green Fluorescent Protein. Cytometry
1999, 37, 51-59.• Brejc, K., Sixma, T. K., Kitts, P. A., Kain, S. R., Tsien, R. Y., Ormo, M., Remington, S. J.
(1997) Discovery and Structure of Green Fluorescent Protein. Proc. Natl. Acad. Sci. U. S. A. 94: 2306–2311
• Campbell, R.E. (2002) History and Discovery of GFP. Proc. Natl. Acad. Sci. USA 99: 7877-7882.
• Moline, M.A. BL Web (2006). Functions of Bioluminescence. Applied env. Microbio. 34: 224-229.
• Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996). Crystal structure of the Aequorea victoria green fluorescent protein. Science, 273:1392-1395
• Ozawa, T., Nogami, S., Sato, M., Ohya, Y., Umezawa, Y.(2000). Analysis of Fluorophore Formation. Anal. Chem.72: 5151-5157.
• Park, S.H., Raines, R. T.(1997). Applications of GFP as HIV Virulence Factor. Protein Science 1997, 6, 2344-2349.