Flourescent protein and its application in cell biology

1. Presented to: Sir Kamran
Presented by: Moheer Fatima
M.Phill pharmacology
FLOUROSCENT PROTEINS

2. 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.

3. 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.

4. 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.

5. 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 .

6. 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.

7. CHARACTERISTICS OF FLOURESCENT
PROTEINS :
 Expressed efficiently
 No phototoxicity
 Bright enough
 Sufficient photostability
 Minimal overlap in excitation and emission profile

8. 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

9. GREEN FLOURESCENT PROTEINS (GFP) :
Green Flourescent proteins
first isolated from the Jellyfish
Aequorea victoria,which lives
in the cold water of pacific
ocean.

10. 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.

11. 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.

13. 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.

14. HOW FLOUROPHORE BECOME ACTIVE OR
MECHANISM OF ACTION:

17. 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.

18. 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

19. 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

20. 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.

21. 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

22. 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

23. EXCITATION AND EMISSION WAVELENGTH:

24. EXCITATION AND EMISSION SPECTRUM:

25. 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,and
understand 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 .

26. GFP WITH HAIRLESS ROOT 1 GENE

27. 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 Phy
B members of phytochrome family of photoreceptors.
Upon irrradiation of red light phy A and phy B will be translocated
to the nucleus.

28. 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.

29. BETA GLUCORONIDASE FUSION WITH GFP

30. 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.
.

31. RNP IN DROSOPHILA EGG CHAMBER

32.  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.

33. • 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

34. 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 .

35. 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 .

37. 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.

38. 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.

39. 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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