Presented to: Sir Kamran

Presented by: Moheer Fatima

M.Phill pharmacology

FLOUROSCENT PROTEINS

FLOURESCENT PROTEINS:

Fluorescent proteins are members of a structurally

homologous class of proteins that share the unique

property of being self-sufficient to form a visible

wavelength flourophore from a sequence of 3 amino

acids within their own polypeptide sequence.

HISTORY:

The presence of a fluorescent component in the bioluminescent

organs of Aequorea victoria jellyfish was noted by Davenport

and Nicol in 1955.

Then,Osamu Shimomura and Frank Johnson, in 1961, first

isolated a calcium-dependent bioluminescent protein from the

Aequorea victoria jellyfish, which

they named aequorin and they

first realize that this fluorophore

was actually a protein.

HISTORY:

About the isolation of the bioluminescent protein

aequorin,Shimomura wrote, "A protein giving solutions that look

slightly greenish in sunlight though only yellowish under

tungsten lights, and exhibiting a very bright greenish

fluorescence in the ultraviolet light.

In 1971 Morin and Hastings isolated very similar green

fluorescent proteins from Obelia .The nature of the

flourorophore itself remained a mystery until 1979 when

Shimomura correctly determined the flourophore to be a 4-(p-

hydroxybenzylidene)-5-imidazolidinone moiety covalently linked

within the polypeptide chain.

INITIAL CLONING AND RECOMBINENT

EXPRESSION :

In 1992 Prasher et al. cloned the gene for GFP from Aequorea

victoria as part of their effort to understand the mechanism of

light generation in the luminescent jellyfish organ

Just two years later came the first dramatic demonstrations that

the gene was self-sufficient to undergo the post-translational

modifications necessary for flourophore formation.

Specifically, Chalfie reported the gene encoding Aequorea

green fluorescent protein could be functionally expressed in the

sensory neurons of the worm Caenorhabditis elegans and

Inouye and Tsuji showed that expression of the gene in

Escherichia coli resulted in green fluorescent bacteria .

WHY DO WE USE FLOURESCENT PROTEINS ???

To track and quantify proteins .

To watch protein protein interaction .

To describe biological events and signal in a cell .

In drug discovery process.

CHARACTERISTICS OF FLOURESCENT

PROTEINS :

Expressed efficiently

No phototoxicity

Bright enough

Sufficient photostability

Minimal overlap in excitation and emission profile

TYPES OF FLOURESCENT PROTEINS:

There are 5 types of Flourescence proteins.

Green Flourescent proteins

Cyan Flourescent proteins

Blue Flourescent Proteins

Yellow Flourescent proteins

Red Flourescent proteins

GREEN FLOURESCENT PROTEINS (GFP) :

Green Flourescent proteins

first isolated from the Jellyfish

Aequorea victoria,which lives

in the cold water of pacific

ocean.

GREEN FLOURESCENT PROTEINS (GFP) :

It produces significant flourescence and is extremely stable, the

excitation maximum is close to the ultraviolet range.

The excitation spectrum of GFP fluorescence has a dominant

maximum at about 400 nm and a significantly smaller maximum

at about 470 nm, while the emission spectrum has a sharp

maximum at about 505 nm and a shoulder around 540 nm .

In addition to enhanced green fluorescent protein, several other

variants are currently being used in live-cell imaging.

STRUCTURE OF GFP:

Composed of 238 amino acids.

The crystal structure of GFP is an eleven-stranded β-

barrel, threaded by an α-helix, running up along the

axis of the cylinder.

The chromophore is in the α-helix, very close to the

centre of the can-like cylinder.

Cylinder has a diameter of about 30A and length is

about 40A long.

Fluorophore located on central helix.

FLOUROPHORE:

The fluorophore itself is a p-hydroxybenzylidene-imidazolidone.

It consists of residues Ser65- dehydroTyr66 –

Gly67 of the protein. The cyclized backbone of

these residues forms the imidazolidone ring.

The fluorescence is not an intrinsic property

of the Ser-Tyr-Gly tripeptide. The amino acid

sequence Ser-Tyr-Gly can be found in a

number of other proteins as well.

This peptide is neither cyclized in any of these,

nor is the tyrosine oxidized. None of these proteins

has the fluorescence of GFP.

HOW FLOUROPHORE BECOME ACTIVE OR

MECHANISM OF ACTION:

LIMITATIONS OF GFP USAGE:

It is generally not well

suited for live cell imaging

with optical microscopy.

slight sensitivity to pH.

weak tendency to

dimerize.

BLUE FLOURESCENT PROTEINS (BFP) :

The blue varients of green fluorescent protein resulted from direct

modification of the tyrosine residue at position 66 (Tyr66) in the

native fluorophore Conversion of this amino acid to histidine results

in blue emission having a wavelength maxima at 450 nanometers.

First used in multicolour imaging and FRET.

Disadvantages:

Excitation of blue proteins is most efficient

in spectral regions that are not commonly

used, so specialized filter sets and laser

sources are required.

Dim

Photobleach easily

CYAN FLOURESCENT PROTEIN (CFP) :

The cyan variants of green fluorescent protein resulted from

direct modification of the tyrosine amino acid to tryptamine results

in a major fluorescence peak

around 480 nanometers along with

a shoulder that peaks around 500

nanometers.

Has a Spectra between BFP and GFP.

Brighter

Display more photostability

Resistant to photobleaching

A Cyan varient have also been introduced termed as

Cerulean.It is 2 fold brighter than CFP.

It is used with yellow fluorescent proteins in FRET

investigations

Disadvantage:

Excitation of blue proteins is most efficient in spectral

regions that are not commonly used, so specialized

filter sets and laser sources are required.

RED FLOURESCENCE PROTEIN (RFP) :

First Red flourescent protein was derived from

• Discosoma striata DsRed

• Heteractis crispa HcRed

Most suitable Red marker.

The fluorescence emission spectrum of DsRed features a peak at

583 nanometers whereas the excitation spectrum has a major

peak at 558 nanometers and a minor peak around 500

nanometers.

Diasadvantage:

DsRed is an obligate Tetramer

DsRed conjugates are toxic

YELLOW FLOURESCENT PROTEINS:

Yellow flourescent protein produced when mutation occur in Threonine residue 203 to Tyrosine.

It show flourescence at 538nm wavelength.

Imaging partner of CFP

(FRET).

Citrine and Venus ,varients

of YFP ,more Brighter than

YFP.

Resistant to photobleaching.

Disadvantages:

Sensitive to acidic pH

EXCITATION AND EMISSION WAVELENGTH:

EXCITATION AND EMISSION SPECTRUM:

APPLICATIONS OF FLOURESCENT PROTEINS:

A. IN PLANTS:

To identify Location of proteins :

To understand how the plant cell is functionally organized,so it is necessary to know where enzymes and regulatory proteins are located in specific plant cells at particular time in development and under particluar environmental conditions .

By fusing GFP Coding sequences to coding regions of genes of unknown location is extremely valuable tool for determining location of protein,andunderstand biochemical or regulatory process,reside within the plant cell.

E.g:

1.GFP/Plant protein fusions localized to the nucleus are the ROOT HAIRLESS

1 gene .

2.Proteins with geranylgeranyl diphosphate synthase activity and NADP-dependent isocitrate dehydrogenase is located in mitochondria .

GFP WITH HAIRLESS ROOT 1 GENE

For identification of Movement of protein:

GFP protein helps in understanding the movement of protein from one compartment to another in plant cells .

Compartments prevent entry of particular proteins ,ions and compunds to prevent undesirable reaction and sequester participants in enzymatic reactions to facilitate cellular processess.

E.g :

GFP sequences are fused to sequences encoding Phy A and PhyB members of phytochrome family of photoreceptors.

Upon irrradiation of red light phy A and phy B will be translocated to the nucleus.

For Identification of compartments:

GFP fusions with transit sequences or entire protein can be used for

deliberate labelling of particular compartment.

The purpose of such experiments may be to study one or more

compartments with regard to number,size ,shape ,mobility,interaction with

other organelles and observation of dynamic changes during development or

environmental response.

E.g:

Formation of chimeric gene by GFP/beta-glucuronidase(GUS) fusion in order

to produce transgenic plants carrying labelled nuclei for studies of nuclear

shape and movement during cell cycle .

Advantage:

By using GFP flourescence as a marker to isolate GFP labelled organelles

and compartments that are not easily separated by more traditional means.

BETA GLUCORONIDASE FUSION WITH GFP

B. IN-VIVO (IN ANIMALS):

In cell and molecular biology:

GFP was first used to look into living cells to monitor protein

localization and to visualize dynamic cellular events .

A fusion between any cloned gene of interest and GFP can be

produced and may be introduced into the organism of interest .

The fate of the resulting protein inside the living cell can be

seen by using flourescence microscopy.

Examples of protein tagging:

1.The first application was tracing of ribonucleoprotein (RNP)

particles trafficking into developing egg chambers of Drosophila.

.

RNP IN DROSOPHILA EGG CHAMBER

2.GFP can be fused to an pre-mRNA splicing factor ,so we can

show the dynamic events That occur inside a cell nucleus during

interphase. We can also see gene expression events such as

transcription.

• An elegant approach developed in Andrew Belmont’s

laboratory,GFP can not only make cellular proteins visible in

living cell ,bt also can make visible DNA sequences.

• E.g

• Robinett et all made use of very tight and specific binding of

bacterial lac repressor protein (lac1) to its DNA target ,the lac

operator (lacO). They introduced repeats of lacO sequence

into the genome of cells and detected the incorporated sites in

living cells using a lacI-GFP fusion protein ,expressed in the

cells of interest.

• Use of this strategy has allowed tracking of in-vivo labelled

DNA sequences in living mammalian,yeast and bacterial cells

over time, and has also led to discovery of a bacterial “Mitotic

apparatus” that is responsible for the equal partitioning of sister

chromosomes during cell devision

Examples of monitoring of gene expression :

It can be used to monitor gene expression in single ,living cells .

GFP gene under the control of any promotor of interest directly indicate the

gene expression level in living cells or tissues.

Has advantage over commonly used expression reporters

E.g:

1.GFP reporter systems are now being used in the development phase of

special purpose vectors such as generation of adenovirus associated virus

based vectors for gene therapy .

Limitations of GFP as a gene reporter:

GFP signal can not be amplified ,so it prevent detection of low expression

level .

Sensitive photon counting devices can overcome this problem ,bt are too

expensive for Routine use .

Examples in genetic screening:

Screening of living cell is specially important in the selection of

embryonic stem cell and production of transgenic animals.

E.g

1.Introduction of GFP into mouse preimplantation embryos and

GFP positive cells selected and used for implantation into foster

mothers to generate transgenic mice .

2.injection of GFP labelled tumor forming cells into nude mice

not only label the tumor .But also allow detection of

micrometastasis in locations distant from the primary tumor .

This mouse model can now be used for the study of tumor

progression .

S

3. Use in drug discovery : To facilitate drug discovery in

the more complex physiological

environment of a cell or organisms

,powerful cellular imaging systems

have been developed.Actually in this

we focus on a single target .These

detection technologies allow analysis

of cellular events and phenotypes.

It also facilitate the integration of

complex biology into the screening process.

4. Use as a biosensors:

GFP is used as a sensor to detect changes or

differences in calcium,pH,voltage,metal and enzyme

activity in a cell .

5. Flourescent proteins are also used in the field of

biophysics ,microbiology and biotechnology.

REFERENCES:

1. Maureen R. Hanson 1 and Rainer H.Kohler ,GFP imaging :Methodology and application to investigate cellular compartmentation in plants,received 31 March 2000; accepted 19 september 2000.

2. J.C.March.G.Rao.W.E.Bentley Biotechnological applications of green flourescent protein ,Received: 23 January 2003 / Revised:7 April 2003 / Accepted : 11 April 2003 / published online : 27 May 2003 Springer –Verlag 2003.

3. Hans-Hermann Gerdes* , Christoph Kaether , Green flourescent protein :application in cell biology ,institute of neurology,university of Heidelberg ,Im Neuenheimer Feld 364, 69120 Heidelberg,Germany received 6 May 1996 .

4. Tom Misteli* and David L.Spector ,Application of the green flourescent protein in cell biology

and biotechnology ,received 9 july 1997;accepted 919 august 1997.

5. Jen Sheen 1*,Seongbin Hwang1, Yasuo Niwa1 ,Hirokazu Kobayashi1,and David W. Galbraith3.Green flourescent protein as a new vital marker in plant cells ,the plant journal 1995 8(5),777-784.

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