Download Green Fluorescent Protein

Green Fluorescent Protein
Avinash Bayya
Varun Maturi
Nikhileswar Reddy Mukkamala
Aravindh Subhramani
Introduction
The Green fluorescent protein (GFP) was first isolated from the Jellyfish Aequorea victoria,
widely found in North America. GFP has existed for more than one hundred and sixty million
years in that species. GFP is a widely studied and highly exploited protein in the fields of
biochemistry and cell biology. The Jellyfish also contains a bioluminescent protein called
aequorin that emits blue light. When GFP is exposed to blue light, or ultraviolet light, it emits
green fluorescent light. Therefore, when the protein aequorin binds with calcium and emits
blue light, this blue light is in turn absorbed by GFP, which then emits bright green light.
GFP has become an amazingly useful protein in scientific research because it allows us to
examine the internal workings of the cell. It is easy to locate the GFP by just exposing it to
ultraviolet light, since it then glows with a bright green fluorescent light. The trick lies in
attaching the GFP to any object that you are interested in examining. For instance, you can
attach the GFP with another protein and examine them through the microscope thanks to
GFP’s fluorescence.
Exploring the Structure
The structure of GFP was solved in 1996 by
Omro et al and Yang et al. Their structures
were included in the PDB with the accession
codes 1EMA and 1GFL, respectively.
GFP consists of 238 amino acids with a
molecular weight of 27 or 30 kD. The protein
fold contains 11 anti-parallel beta strands and
alpha helices. These strands and helices form
a classical beta barrel structure. (Figure 1),
enclosing the Ser-Tyr-Gly (chromophore)
residues in order to protect them from
solvent interactions. Thus, GFP is often
referred to as “Light in the can”.
Chromophore
The chromophore is formed by p-hydroxybenzylideneimidazolinone formed from residues
65-67, which are Ser-Tyr-Gly of the native protein. The green shade in Figure 2 indicates the
structure of the chromophore.
The mechanism for the
formation of the chromophore
takes place in four steps;
folding, cyclization, dehyration
and oxidation. First the
denatured GFP is folded to
form the native conformation
with a half time of 10 min. This
is followed by the cyclization,
where the imidazolinone is
formed
by
nucleophilic
interaction between the amide
of Gly67 and the carbonyl of
residue
Ser65,
and
the
dehydration.
Then,
the
molecular
oxygen
dehydrogenates the bonds of
the aromatic group of residue
66 in conjunction with
imidazolinone (Figure 3). At
this stage the chromophore is
stable and can acquire visible
absorbance and fluorescence.
In the chromophore there is a special interaction between glycine and threonine creating a
new bond, which in turn creates an unusual five-membered ring (Figure 4).
Figure 4. Chromophore illustrated in SwissPDBViewer.
GFP, when viewed at high resolution, offers great explanation between the protein structure
and spectroscopic functions. The chromophore is situated in the middle of the GFP, totally
protected from the environment. This shielding is essential for the fluorescence. When the
chromophore absorbs a photon, jostling water molecule would take away the energy of the
chromophore. But inside the protein, the chromophore is protected by releasing a less
energetic photon of light, instead of releasing energy. Some important polar groups buried
beside the chromophore are Gln69, Arg96, His148, Thr203, Ser205 and Glu222. Also, GFP’s
dimeric form is highly influenced by the compulsory conditions for its expression.
Changes in GFP results in the release of new colors
Substitutions of the amino acids in the chromophore region results in change of GFP color
when exposed to blue or ultraviolet light. There are three GFP variants; blue fluorescent
protein (BFP), yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). The
amino acid substitutions in the GFP variants are shown below (Table 1).
Table 1. Amino acid substitutions in the GFP variants.
Amino acids
Color
GFP
Green
65
66
67
68
69
72
145
146
153
163
203
wtGFP Phe
Ser
Tyr
Gly
Val
Gln
Ser
Tyr
Asn
Met
Val
Thr
Green
EGFP
Leu
Thr
Blue
EBFP
Leu
Thr
Yellow EYFP
Phe
Gly
Cyan
Leu
Thr
ECFP
64
His
Phe
Leu
Trp
Ala
Tyr
Ile
Thr
Ala
The above table represents the position of the amino acid in various GFP variants. These GFP
variants are more enhanced than the wild type GFP and the result is that more fluorescence
can be seen in mammalians. These variants can be used as reporter genes in order to learn
about protein interactions, timing of cell cycle events, and much more.
GFP as a biosensor
GFP is used within various fields of biology like molecular biology, neuroscience and cell
biology. It can be used as a reporter gene, cell marker, fusion tag, and for quantitative
monitoring of gene expression. GFP’s fluorescence characteristic can also be used in methods
designed for sensing various levels of ions or pH. As mentioned above, there have been many
codon alterations performed in GFP in order to make the expression effective in mammalians.
As an application in genetic engineering, fluorescent green rabbits, rats, mice, frogs, flies,
worms, and other living organisms have been produced.
Below, the use of GFP as a biosensor for measuring the concentration of lead in aquatic
environments will be described. Here, the GFP gene is under the control of the lead resistance
regulatory gene (PbrR) and its operator promoter (PbrO/P), originating from the plasmid
pMOL30 of Ralstonia metallidurans. The regulatory gene and its promotor were isolated by
PCR using the PbrR/O/P forward and reverse primers carrying Nhe1 and Age1 sites,
respectively. The amplified PCR product was cloned into the pDB402 plasmid containing the
gfp gene. The entire PbrR/O/P/GFP was excised using Nhe1 and EcoRV and inserted into the
pBR322 vector, and transformed into E. coli DH5α cells. A correctly cloned and confirmed
sequence was named the
Pbr-GFP
biosensor
(Figure 5).
The Pbr–GFP biosensor
was grown in LB broth
with
100
µg/ml
ampicillin at 37OC, and
streaked on to LB-amp
plates with various
concentrations of lead
ions (Pb2+). The plates
were incubated at 37OC
for
16h.
The
fluorescence was then
directly measured using
the Zeiss florescence
stereozoom microscope
at 25× at an excitation
range of 450-490 nm
and an emission range of
500-550 nm.
The expression of GFP was evaluated at the following Pb2+ concentrations: 50, 100, 150, 200,
250, 300, 350, and 400 µM. The fluorescence showed a steady increase from 50 µM and
upwards, peaked at 250 µM, and then again decreased. The fluorescence was also highest
after 12 h. The following formula (based on the 12 h measurements) was derived using
multiple regressions, and it can be used in order to estimate the Pb2+ concentration in an
unknown water sample with 95% accuracy.
C = (1629.02458 + (-0.00010809×D) + (0.00000502×F)
Where, C: Concentration of Pb2+ in the sample
D: Cell density
F: GFP fluorescence in terms of RLU (Relative Light Unit)
1629.02458, -0.00010809 and 0.00000502 are constants.
Discussion
GFP can be used as a reporter gene; it shows high sensitivity, it is non-toxic, time efficient
and cost effective when compared to the other classical reporter genes like lacZ and GUS.
Many spectral variants of GFP are now available, and therefore it is possible to label different
proteins, in different colors, inside the same cell. GFP can also be used as an active tool in
finding the dynamics of chromosomes in vivo. It can be used in the studies of active bacterial
chromosome segregations, yeast mitosis, and centromere dynamics, by tagging it to specific
chromosomes. As a last example, GFP can be used as a biosensor for detecting the lead ion
concentration in water. It is easy to use this technique to estimate the concentration of any
simple substance in the water that can be grown in media.
References
•
Virapong Prachayasittikul, Chartchalerm Isarankura-Na-Ayudhya, Yaneenart
Suwanwong, Srisurang Tantimavanich., Construction of chimeric antibody binding
green fluorescent protein for clinical application, EXCLI Journal 2005;4:91-104.
Tapas Chakraborty, P. Gireesh Babu, Absar Alam and Aparna Chaudhari., GFP
expressing bacterial biosensor to measure lead contamination in aquatic environment,
CURRENT SCIENCE, VOL. 94, NO. 6, 25 MARCH 2008.
Roger Y. Tsien., Annu. Rev. Biochem. 1998. 67:509–44
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http://www.cryst.bbk.ac.uk/PPS2/projects/jonda/intro.htm
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http://www.ebbep.org/docs/pglo/gfpstructure.pdf
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http://www.clontech.com/images/pt/PT2040-1.pdf
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http://www.conncoll.edu/ccacad/zimmer/GFP-ww/structure.html
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http://www.cryst.bbk.ac.uk/PPS2/projects/jonda/chromoph.htm
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http://www.pdb.org/pdb/explore/images.do?structureId=1EMA
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
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