Autofluorescence - natural emission of light by biological structures  when they have absorbed light. The most commonly observed autofluorescencing molecules are NADPG and flavins. Generally, proteins containing an increased amount of the amino acids tryptophan, tyrosine and phenylalanine show some degree of autofluorescence. To overcome cons of labeling for in-vivo applications mostly, naturally fluorescent proteins are used.

1. PRESENTEDTO:
DEPARTMENTOF CHEMISTRY
PRESENTEDBY:
VIDUSHI SHARMA
M.TECH 1ST YEAR
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2. CONTENTS:
I. INTRODUCTION
i. Auto-fluorescent Proteins
ii. Biosensors
II. STRUCTURE- SPECTRA RELATIONSHIP OF GFP
III. FRET CONCEPT
IV. GFP IN BIOSENORS
i. Invivo biosensors
ii. Invitro biosensors
V. ADVANTAGES OF GFPAS FLUROSCENT MARKERS
VI. OTHER APPLICATIONS OF GFP
VII.FUTURE ASPECT
VIII.CONCLUSION
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3. i. Auto-fluorescent Proteins
•Autofluorescence - natural emission of light by biological structures when they
have absorbed light
•The most commonly observed autofluorescencing molecules
are NADPG and flavins
•Generally, proteins containing an increased amount of the amino acids
tryptophan, tyrosine and phenylalanine show some degree of autofluorescence.
•To overcome cons of labeling for in-vivo applications mostly, naturally fluorescent
proteins are used.
•Genetically encode fluorescent tags
3

4. Green Fluorescent Protein (GFP)
•Jellyfish Aequerea victoria
• Discovered in 1955 by Davenport and
Nichol
•Shimomura in 1966-
•Protein of 238 amino acids,
•Bright green fluorescence when exposed to
light in blue to ultraviolet region
•Quantum Yield of 0.79
•2 excitation peaks: 395nm and 475nm
•Emission peak- 509nm
Figure 1: Green emission from jellyfish.
Green fluorescent protein is a protein
extracted from jellyfish, which fluoresces
green when placed under blue light. Source:
sunsetwiki.brown.edu
Dobbie et al, “Autofluorescent proteins” Methods cell Biology, 2008;85:1-22.
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5. DsRed- Red Fluorescent Protein
•Discosoma striata
•28 kDalton protein
•Excitation- 560nm
•Emission- 580nm
•Negligible ph dependence
•Photobleaching resistant
Figure 2: Discosoma striata, non-bioluminescence corals
source of DsRed.
Source: http://chemistry.berea.edu/~biochemistry/2008/om/
1) Dobbie et al, “Autofluorescent proteins” Methods cell Biology, 2008;85:1-22
2) Larrainzial et al, “Applications of Autofluorescent Proteins for In Situ Studies
in Microbial Ecology”, Annu. Rev. Microbiol. 2005.59:257-277
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6. ii. Biosensors
•Biosensor is an analytical device combining a sensitive biological element
such as microorganism, antibody, cell receptors, enzymes etc, with a
physicochemical detector.
•This sensitive biological element is attached to a transducer or detector
element which generates a signal which can be measured and quantified on
basis of interactions of analyte with sensor.
•Bioreceptors interacts with the analyte.
Figure 3: Schematic of a Biosensor. Source:
www.rpi.edu
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7. Sequences of various autofluorescent proteins. It shows sequences might
vary, however few amino acids are conserved at their position since they are
responsible for secondary structure which is highly conserved in all these
proteins. Dobbie et al, “Autofluorescent proteins” Methods cell Biology,
2008;85:1-22
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8. Figure 4: The central chromophore, a p-hydroxybenzylideneimidazolinone,
formed by the cyclization of the core amino acids, Ser65, Tyr66, and Gly67.
Dobbie et al, “Autofluorescent proteins” Methods cell Biology, 2008;85:1-22
First, GFP folds into a nearly native conformation, then the imidazolinone is
formed by nucleophillic attack of the amide of Gly67 on the carbonyl of residue
65, followed by dehydration.
Finally, molecular oxygen dehydrogenates the α-β bond of residue 66 to put its
aromatic group into conjugation with the imidazolinone.
Only at this stage does the chromophore acquire visible absorbance and
fluorescence. Tsein, “THE GREEN FLUORESCENT PROTEIN”, Annu. Rev.
Biochem. 1998. 67:509–44
8

9. 3 Major steps in formation of a chromophore in GFP during folding:
1. Cyclization
2. Dehydration
3. Oxidation
Mechanism is based upon arguments:
• Atmospheric oxygen is required
• In unaerobic GFP, fluorescence develops only after oxygen is re-admitted
• Analogous imidazolinones autoxidized spontaneously
• Gly 67 is always conserved in all mutants as it acts as a good nucleophile
in cyclization because of minimal stearic hindrance
• Hydrogen peroxide released can be harmful
Mechanism proposed for chromophore synthesis in GFP. Tsein, “THE GREEN
FLUORESCENT PROTEIN”, Annu. Rev. Biochem. 1998. 67:509–44
9

10. Mechanism proposed for chromophore synthesis in GFP. Tsein, “THE GREEN
FLUORESCENT PROTEIN”, Annu. Rev. Biochem. 1998. 67:509–44
10

11. Secondary and tertiary structures:
Figure 5: β barrel structure of DsRed conserved as in all other AFPs such as wild type GFP,
containing helix and a chromophore in center. Source: zeiss-campus.magnet.fsu.edu
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12. •Multiple folding pathways
•11 stranded beta barrel anti-parallel strands, each of 9-13 residues in length
•H-bonds with adjacent strands to form closed structure
•Bottom terminal has 2 distorted helical crossover segments
•Top has one short crossover distorted segment
•Fluorophore is burried as distorted alpha helix in interior of barrel
•Residues facing barrel center effect fluorescence properties
•Interior cavity is slightly polar with 4 water molecules on one side of alpha helix
while other side has hydrophobic residues
•Polar side chains to stabilize chromophore: His 148, Thr-203, Ser205, form H-
bonds with Phenolic group of chromophore
•Arg96 and Gln94- interact with carboxylic group of imidazolide and form salt
bridges with negatively charged oxygen atom
•Difusibility of oxygen in barrel center essential
•Coplanar aromatic rings of the chromophore adopt cis conformation across
Tyr66 alpha-beta double bond
State of art in biosensor, Chapter: GFP based biosensors by Crone et al.
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13. FACTORS AFFECTING FLUORESCENCE:
•Residues facing center of barrel influence fluorescenece properties such as
spectra, maturation time, photostability
•Rate of folding
•Fluorophore formation
•Propensity of multimerization
•Quenched by water and oxygen interaction in unfolded state
•Ph dependence of spectral absorbance due to protonation of chromophore
1. Dobbie et al, “Autofluorescent proteins” Methods cell Biology, 2008;85:1-22
2. State of art in biosensor, Chapter: GFP based biosensors by Crone et al.
3. Mechanism proposed for chromophore synthesis in GFP. Tsein, “THE GREEN
FLUORESCENT PROTEIN”, Annu. Rev. Biochem. 1998. 67:509–44
13

14. 14

15. Fluorescence Resonance Energy Tranfer
The mechanism of fluorescence resonance energy transfer involves a donor
fluorophore in an excited electronic state, which may transfer its excitation
energy to a nearby acceptor chromophore in a non-radiative fashion through
long-range dipole-dipole interactions. The theory supporting energy transfer is
based on the concept of treating an excited fluorophore as an oscillating dipole
that can undergo an energy exchange with a second dipole having a similar
resonance frequency.
Syed Arshad Hussain, “An Introduction to Fluorescence Resonance Energy Transfer (FRET)”,
Science Journal of Physics, August 2012, Volume 2012, ISSN:2276-6367
15

16. Figure 7: Jabolski Diagram for FRET process and Absorption and fluorescence spectra of an ideal
donor-acceptor pair. Brown coloured region is the spectral overlap between the fluorescence spectrum
of donor and absorption spectrum of acceptor. Source: Syed Arshad Hussain, “An Introduction to
Fluorescence Resonance Energy Transfer (FRET)”, Science Journal of Physics, August 2012, Volume
2012, ISSN:2276-6367
16

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Group 1: Intramolecular FRET based
biosensor
Group 2: Intermolecular FRET based
biosensor
Group 3: BiFC based biosensor
Group 4: Single FP based exogenous
MRE biosensor
Group 5: Single FP based endogenous
MRE biosensor
Ibraheem et al, “Designs and applications of fluorescent protein-based biosensors”, Current Opinion in Chemical
Biology 2010, 14:30–36

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Figure: Schematic model of a generic intramolecular FRET-based biosensor. A FP FRET pair
flanks an MRE that undergoes a conformational change that alters the distance and/or
orientation of the FPs relative to each other. An MRE suitable for the detection of protease
activity, detection of PTM enzymatic activities where the modification of the peptide substrate
creates a binding dock for the binding domain resulting in a FRET change. Source: Ibraheem et
al, “Designs and applications of fluorescent protein-based biosensors”, Current Opinion in
Chemical Biology 2010, 14:30–36

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Group 2: Intermolecular FRET
based biosensor
Group 3: BiFC based
biosensor
Group 4: Single FP based
exogenous MRE biosensor
Group 5: Single FP based
endogenous biosensor

20. IN VIVO BIOSENSORS
1. Ph biosensors
2. Enzyme activity biosensors
3. Detection of ROS and antioxidant
4. Calcium ion detection
IN VITRO BIOSENSORS
1. GFP antibody chimeric protein
2. Chimeric biosensor based on allostery
3. FRET Based using Quantum Dots
4. Intrinsic Ion sensor
1. State of art in biosensor, Chapter: GFP based biosensors by Crone et al.
2. Ibraheem et al, “Designs and applications of fluorescent protein-based biosensors”, Current Opinion
in Chemical Biology 2010, 14:30–36
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21. IN VIVO BIOSENSOR: PH CHANGES
Sensitivity to pH which results from protonation and deprotonation of the chromophore
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22. IN VIVO BIOSENSOR: Enzymatic activity
Figure 10 and 11: Arrestin protein conjugated in GFP to monitor GFCR activation and GPCR- C protein coupled receptor.
A and B images shows Presence of kinase and its activity near plasma membrane in a cell. Larry et al, “A b-Arrestin/Green
Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation”, THE JOURNAL OF BIOLOGICAL
CHEMISTRY, Vol. 272, No. 44, Issue of October 31, pp. 27497–27500, 1997
Figure 9: FRET based sensors are constructed so that the substrate protein of the kinase of interest is flanked with a
fluorescent protein pair in such a way that the conformational change imparted by phosphorylation translates into a
change in the FRET signal. Source State of art in biosensor, Chapter: GFP based biosensors by Crone et al.
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23. IN VIVO BIOSENSOR: CALCIUM ION SENSOR
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Figure 12 : Comparison of a single FP-based (A) and a FRET-based (B) genetically-encoded Ca2+ sensor. (A) GCamP consists
of a circularly permutated GFP in which calmodulin (CaM) and M13 are grafted into the beta-barrel using the newly
available N- and C-termini. In absence of Ca2+, the FP is only dimly fluorescent, due to solvent quenching of the fluorophore.
Ca2+ binding and the concomitantly induced M13-calmodulin interaction excludes solvent from the chromophore,
dramatically increasing fluorescence intensity. The graph on the right shows the typical change in emission spectrum of
GCaMP and related sensors upon Ca2+ addition. (B) The Cameleon FRET sensor for Ca2+ also uses CaM and M13, but now
sandwiched between CFP and YFP. The Ca2+-binding-induced intramolecular CaM-M13 interaction results in an increase in
FRET. The graph on the right shows the typical emission spectra in presence and absence of Ca2+ seen with Cameleon and
related sensors. Source: Laurens Lindenburg and Maarten Merkx , “Engineering Genetically Encoded FRET Sensors”,
Sensors 2014, 14(7), 11691-11713; doi:10.3390/s140711691

24. IN VITRO BIOSENSOR: GFP based antibody
For the same kind of nanomolar sensitivity as that of antibodies, two antigen-
binding loops are inserted into the GFP structure: regions β4/β5 (residue 102),
β7/β8 (residue 172) and β8/β9 (residue 157).
Studies have revealed several mutations that conferred additional stability and
increased fluorescence in the context of inserted loops: D19N, F64L, A87T,
Y39H, V163A, L221V, and N105T
Source State of art in biosensor, Chapter: GFP based biosensors by Crone et al.
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Antibody
Locating
molecules
Secondary
antibody
Detection
Allostry
GFP
Biosensor

25. IN VITRO BIOSENSOR: A chimeric fluorescent biosensor based
on allostery
25
Receptor
Inserted into
GFP
Ligand
To be
detected
Conformational change in
GFP leading to change in
emission or quenching
Studies were performed and receptor Bla1 was coded in loop for ligand BLIP
successfully. Source : State of art in biosensor, Chapter: GFP based
biosensors by Crone et al.

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IN VITRO SENSOR: FRET BASED USING QUANTUM DOTS
Figure 13 : Showing emission of the chromophore only when in close proximity to the QD. When the
two are split by caspase activity, FRET is lost. The imidazole side chains of the histidines electronically
coordinate with the zinc atoms of the CdSe—ZnS core-shell semiconductor of the QD. Multiple
mCherry molecules can be coordinated with each QD. Seperation of mCherry from the QD by a
protease will lead to loss of FRET. By placing the caspase-3 cleavage sequence into the linker between
GFP and the QD, the FRET complex becomes a biosensor for the presence of caspase-3, glowing red at
610 nm in the absence of the protease, and reverting to the yellow fluorescence of the QD at 550 nm
when the protease is present. Source : State of art in biosensor, Chapter: GFP based biosensors by
Crone et al.

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IN VITRO BIOSENSOR: INTRINSIC ION SENSOR
To make GFP
sensitive to
Ion
concentrations
Mutations for
affinity
Protonation/
deprotonation
Ion channels
to enter barrel
and quench
chromophore
Figure 14: The analyte channel
through which copper ions can pass
through to the interior of the barrel
structure and quench the fluorescence
of the chromophore. Source : State of art
in biosensor, Chapter: GFP based biosensors
by Crone et al.

28. Multiple labeling of proteins possible
Biocompatible
Dynamic labeling possible for in vivo processes
Signals can be modifies as per requirements via amino acid mutations
Feasible for in vivo as well as in vitro processes
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More AFPs to discover in other
species
Many multimeric proteins converted
to monomeric proteins
Nanometer resolution
Development of biosensors of
numerous other dynamic processes

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Its been many decades since AFPs have been discovered, yet a
lot of information about their spectra- structure relationship
and expression are yet to be explored. With huge applications in
in vivo as well as in vitro studies as markers and sensors, they
hold capacity to revolutionize the face of science and technology
in genre of medical sciences, chemistry , biotechnology and
nano-technology

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