1. Fluorescent proteins and their applications
Fluorescence principles
The Green Fluorescent Protein
The DsRed Fluorescent Protein
Applications of fluorescent proteins

2. Molecular or atomic orbital energy levels
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
First, a few words about fluorescence.
The fluorophore absorbs photons in the excitation spectrum. Part of the energy of the absorbed photon is lost in a non-radiative manner, and the rest is emitted as a photon in the emission spectrum. The difference in energy between the emission and excitation spectrum maxima is called the Stockes shift and corresponds to the energy lost in a non-radiative manner. The quantum yield is the number of photons emitted divided by the number of photons absorbed.
emission spectrum: lexcitation fixed; lemission variable
excitation spectrum: lemission fixed; lexcitation variable

3. Jablonski diagram Absorption spectrum Fluorescence spectra 2 E2
2 : non-radiative transition
3 : emission 1 3 E1 2
Absorption spectrum
Fluorescence spectra
Fluorescence intensity
O.D.
absorption
excitation
emission
The Jablonski diagram is representative of the energy transitions that take place in fluorescent molecules.
The electronic energy levels are represented as E1 and E2. The sub-levels correspond to vibrational levels. At room temperature, the lowest energy level is the most populated because the vibrational energy is comparable to a few kBT.
Absorption of an excitation photon, and therefore the excitation spectrum, corresponds to the transition between the lowest energy level in E1 and any energy level in E2 (1). During the next nanosec, the electron relaxes to the lowest energy level of E2, transferring the vibrational energy to the solvent (2). A fluorescence photon is then emitted, that corresponds to the transition between the lowest energy level in E2 and any energy level of E1 (3). The electron then relaxes to the lowest energy level (4). This mechanisms explains that the excitation and the emission spectra are symmetrical. The difference between there maxima, or Stockes shift, corresponds to the energy lost in a non-radiative manner. The excitation spectrum matches the peaks in the absorption spectrum that give rise to a fluorescence emission.
absorption
wavelength
excitation
emission
wavelength
Stockes shift

4. Luminescence of aequorin and GFP
Discovery of Aequorin and the Green Fluorescent Protein (GFP)
Aequorea victoria
PDB structure : 1EMA
Aequoria victoria GFP
Luminescence of aequorin and GFP
The first fluorescent protein was discovered in a jellyfish called Aequorea victoria (top left). The jellyfish is transparent, but possesses a series of light emitting organs underneath the bell margin, that emit flashes of blue-greenish light in order to attract preys. Two proteins are involved in light emission, aequorin and the green fluorescent protein.
In the presence of Ca2+, aequorin catalyzes the reaction : coelenterazine, + O2  coelenteramide + CO2.Coelenteramide is unstable and emits blue light (469 nm). The Green Fluorescent Protein (GFP) absorbs the blue light and emits green light (509 nm). Intracellular releases of Ca2+ control the light emission.
The GFP structure is shown on the right. The protein consists of a beta-barrel, a cylindrical structure formed by a rolled beta sheet, surrounding an alpha helix containing the fluorophore. The fluorophore is made by the three consecutive amino acids SYG, that undergo a post-translational modification explained below. The fluorophore is rather well protected from the aqueous medium, which ensures a high quantum yield (0.8) and a a good stability.

5. Niwa, Haruki et al. (1996) Proc. Natl. Acad. Sci. USA 93, 13617-13622
The GFP fluorophore
cyclization
GFP is a 238 amino acid long protein. The fluorophore is made by the three consecutive amino acids S65YG67, which undergo two chemical reactions represented in panel B. The presence of O2 is therefore required for the GFP to become fluorescent. The fluorophore contains alternating double and simple bonds (polyene) that can delocalize and thus allow an electronic transition. The absorption energy depends on the number of delocalized double bonds. The largest the number of double bonds, the highest the wavelength.
In addition, the GFP fluorophore can bind a proton, which changes the light emission properties of the molecule. This pH sensitivity is usually unwanted and Roger Tsien improved the GFP by introducing a single point mutation (S65T), which increases fluorescence emission, improves photostability, and makes GFP less sensitive to pH.
Blue fluorescent protein (BFP) and cyan fluorescent protein (CFP) were obtained by introducing the Y66H and Y66W mutations, respectively. These mutations reduce the number of delocalized double bonds in the fluorophore. The yellow fluorescent protein (YFP) was obtained by introducing the mutation T203Y. The red shift is due to π-electron stacking interactions between the substituted tyrosine residue and the fluorophore.
oxidation
pH sensitivity
Niwa, Haruki et al. (1996) Proc. Natl. Acad. Sci. USA 93,

6. Discovery of Red Fluorescent Proteins
PDB structure : G7K
Discosoma sp DsRed protein
Discosoma reef coral
A red fluorescent protein was discovered in an coral of the Discosoma family. The endogenous protein is structurally related to GFP, but the fluorophore is different (see next slide), the protein is tetrameric and it undergoes a complex and lengthy maturation.
Genetic engineering allowed Robert Tsien and his colleague to design a set of fluorescent proteins that are monomeric, that maturate faster and that emit from the yellow to the infrared. The red fluorescent protein is usually called DsRed, which refers to the organisms where it was discovered.
Red fluorescence
Long maturation time
Tetrameric protein
 Directed mutations : set of yellow to far-red fluorescent proteins

7. DsRed structure green fluorescence DsRed chromophore : QYG
Auto-oxidative fluorophore generation
green fluorescence
DsRed chromophore : QYG
blocked in K83R mutant
DsRed is structurally similar to GFP, as shown by the superposition of the polypeptide backbones in the left panel. The DsRed fluorophore is QYG, which undergo two successive oxidations. The first one creates a green fluorescent protein, and the second one extends the number of delocalized double bonds, which shift the absorption spectrum to the red.
From DsRed, several proteins have been engineered, that can be photoactivated (a flash of light converts them from the GFP form to the RFP form). Further readings are available in
- Lippincott-Schwartz J, Patterson GH. (2008) Fluorescent proteins for photoactivation experiments. Methods Cell Biol.;85:45-61.
- Bourgeois D, Adam V. (2012) Reversible photoswitching in fluorescent proteins: a mechanistic view. IUBMB Life. 64:482-91
Comparison of GFP and DsRed polypeptide structure
red fluorescence
Yarbrough & al. 2001
Proc. Nat. Ac. Sci. 98 :
Ser65
GFP chromophore : SYG

8. Visualization of proteins of interest, in fusion with fluorescent proteins
Protein of interest
codon 3
codon 2
GFP
codon 2
codon 3
ATG
stop
promoter
Fluorescent proteins are used to visualize proteins of interest, in cells and in organisms. Mice expressing GFP and exposed to blue light are shown in the top panel.
The bottom panel displays the different colors of the most commonly used fluorescent proteins. The right panel represents the structure of a typical plasmid used to express a protein of interest in fusion with a fluorescent protein. This plasmid can then be expressed in a cell line in order to visualize the localization of the fusion protein by optical microscopy. Examples of such plasmids will be used in the cell biology labworks.
383
445
439
476
548
562
587
610
590
649
Nathan Shaner et al (2004) Nature Biotech. 22:
Lei Wang et al (2004) Proc. Natl. Acad. Sci. USA 101:
From R. Tsien NP lecture

9. 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
Place and time where a promoter is active
Transcription factor activity
Labeling of specific cells in an organism
GFP
Polypeptide whose conformation is sensitive to a molecule of interest
In vivo measure of the concentration of a molecule of interest (FRET, FLIM)
Promoter
In this slide, several reporter constructs are represented, that are commonly used to study protein function in living cells.
The first construct consists of a protein of interest fused in frame with a fluorescent protein. It is used to monitor the expression and localization of a protein of interest.
The second construct consists of a promoter of interest fused with a fluorescent protein. It is used to monitor when and where the promoter is active, in a cell or an organism.
The third and fourth constructs consist of two fluorescent proteins separated by a polypeptide whose conformation can change, depending of the presence of a molecule of interest, or upon phosphorylation by a kinase. The distance between the two fluorescent proteins can change, which gives rise to FRET or FLIM signals.
FRET, or fluorescent resonant energy transfer, is a non-radiative energy transfer between two fluorophores. Instead of emitting blue fluorescence, the energy absorbed by the CFP is transferred to the YFP that emits yellow fluorescence. The FRET signal is the yellow/blue emission ratio.
FLIM, or fluoresence lifetime imaging, is a microscopy technique where the lifetime of the fluorescence emission is measured at each pixel. FRET induces a change of the fluorescence lifetime of the fluorophore that can be measured to determine the proportion of different conformational states of the reporter molecule.
CFP
YFP
Polypeptide able to bind the phosphorylated form of the substrate
In vivo measure of the activity of a kinase of interest (FRET, FLIM)
Kinase substrate
Promoter
CFP
YFP

10. Osamu Shimomura Martin Chalfie Robert Tsien Douglas C. Prasher
NP 2008 for the discovery of GFP and aequorin
Martin Chalfie
NP 2008 for the first use of GFP as a reporter in the nematode
Robert Tsien
NP 2008 for the development of intracellular fluorescent indicators, especially for Ca2+
The last slide represents different scientists who made important contributions to the fluorescent protein field.
Osamu Shimomura studied Aq. victoria luminescence and discovered aequorin and GFP.
Martin Chalfie used GFP as a reporter gene in Caenorhabditis elegans, a small worm which is a good experimental model of multicellular development.
Robert Tsien engineered many fluorescent proteins, and fluorescent reporters.
Their scientific work was acknowledged by the 2008 Nobel Prize.
Douglas C. Prasher cloned the GFP gene, determined its sequence in 1992 and distributed the plasmid to many scientists. Unfortunately, his research grant was not renewed, and he was forced to leave science. At the Noble Prize conference, M Chalfie and R Tsien acknowledged the essential contribution of D. C. Prasher to their own work. Heis now working in Roger Tsien's lab at the University of California in San Diego (Wikipedia).
Douglas C. Prasher
Cloned in 1992 the GFP gene (Gene, 111 (1992) )