Fluorescent proteins and their applications
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Fluorescence principles
The Green Fluorescent Protein
The DsRed Fluorescent Protein
Applications of fluorescent proteins
1
Excitation and emission spectra of fluorophores
Molecular or atomic
orbital energy levels
Non-radiative
transition
Eexcitation
= hc/excitation
Eemission
= hc/émission
Non-radiative
transition
Resting state
Excited state
 emission spectrum: excitation fixed; emission variable
 excitation spectrum: emission fixed; excitation variable
2
 Jablonski diagram
2
E2
1 : absorption
1
E1
3
3 : emission
2
 Absorption spectrum
O.D.
absorption
 Fluorescence spectra
Fluorescence
intensity
absorption
wavelength
2 : non-radiative transition
excitation
emission
excitation emission wavelength
Stockes
shift
3
Discovery of Aequorin and the Green Fluorescent Protein (GFP)
PDB structure : 1EMA
Aequoria victoria GFP
Aequorea victoria
Luminescence of
aequorin and GFP
4
The GFP fluorophore
cyclization
oxidation
pH sensitivity
Niwa, Haruki et al. (1996) Proc. Natl. Acad. Sci. USA 93, 13617-13622
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Discovery of Red Fluorescent Proteins
Discosoma reef coral
PDB structure : G7K
Discosoma sp DsRed protein
http://www.reefpedia.com/index.php/Discosoma
• Red fluorescence
• Long maturation time
• Tetrameric protein
 Directed mutations : set of
yellow to far-red fluorescent
proteins
6
DsRed structure
Auto-oxidative fluorophore generation
green
fluorescence
DsRed chromophore : QYG
blocked
in K83R
mutant
Comparison of GFP and DsRed
polypeptide structure
Yarbrough & al. 2001
Proc. Nat. Ac. Sci. 98 : 462-67
red
fluorescence
Ser65
GFP chromophore : SYG
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Visualization of proteins of interest, in fusion with fluorescent
proteins
383 439
445 476
548
562
587
610
590
649
Nathan Shaner et al (2004) Nature Biotech. 22:
1567-1572
Lei Wang et al (2004) Proc. Natl. Acad. Sci. USA
101: 16745-16749
From R. Tsien NP lecture
8
Reporter genes
A DNA of interest can be integrated in the genome of cells or organisms
Protein of interest
GFP
Localization of a protein of interest
Promoter
of interest
Promoter
Promoter
Place and time where a promoter is active
Transcription factor activity
Labeling of specific cells in an organism
GFP
CFP
CFP
Polypeptide whose
conformation is
sensitive to a
molecule of interest
In vivo measure of the
concentration of a
molecule of interest
(FRET, FLIM)
YFP
Polypeptide able
to bind the
phosphorylated
Kinase form of the
substrate substrate
YFP
In vivo measure of
the activity of a
kinase of interest
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(FRET, FLIM)
Osamu Shimomura
Martin Chalfie
Robert Tsien
NP 2008 for the
discovery of GFP and
aequorin
NP 2008 for the first use of
GFP as a reporter in the
nematode
NP 2008 for the development
of intracellular fluorescent
indicators, especially for Ca2+
Douglas C. Prasher
Cloned in 1992 the GFP
gene (Gene, 111 (1992)
229-233 )
http://www.telegraph.co.uk/news/worl
dnews/northamerica/usa/3178845/The
-scientist-the-jellyfish-protein-and-theNobel-prize-that-got-away.html#
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