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.