Institut de Biologie StructuraleGrenoble / France

Photophysics of fluorescent proteins

Photophysics of fluorescent proteins

Alexandre Jablonski, a Polish physicist, proposed in 1935 a diagram to explain the mechanism of fluorescence.

A fluorescent molecule is able to absorb light energy (excitation) and emit rapidly fluorescence (emission). From the excited state S1, other pathways for relaxation exist, such as radiationless decay via internal conversion or inter-system crossing to a triplet state, which may subsequently relax via phosphorescence or chemical reactions. Fluorescence emission occurs from the vibrational minimum of S1 to S0. Return to the S0 state can also occur through interaction with a second molecule through fluorescence quenching. Molecular oxygen (O2) is an extremely efficient quencher of the triplet state because of its unusual triplet ground state.

In the years 1960-70, GFP (Green Fluorescent Protein) has been purified from jellyfish Aequorea Victoria and studied by Osamu Shimomura. In 1992, Douglas Prasher was successful in cloning the wild-type GFP gene, and in 1994, Martin Chalfie expressed the coding sequence in E. coli and C. elegans cells. Then, in the beginning of the 21st century, Roger Tsien engineered GFP to develop variants of multiple colors. In 2008, Shimomura, Chalfie and Tsien got the Nobel Prize in Chemistry for these achievements. The first crystallographic structure of GFP has been solved by James Remington in 1996. GFP has a molecular mass of about 27 kDa and is composed of 238 amino acid residues. GFP adopts a β-barrel structure with a diameter of 24 Å and a length of 42 Å. This cylinder-shaped protein is composed of 11 (mostly) antiparallel β strands (1 and 6 are parallel) and an α helix through its center containing the chromophore that is formed autocatalytically from residues 64-66 . This structure, largely conserved within all FPs, maintains the chromophore in a relatively rigid conformation promoting fluorescence, and protects it to some extent from solvent interactions.

GFP has a major excitation band at 395 nm (protonated chromophore) and a minor excitation band at 470 nm (deprotonated chromophore). Excitation at both bands results in green fluorescence emission at 509 nm.

It is important to note that the protonated chromophore hardly fluoresces. Upon excitation at 395 nm, the protonated residue Tyr66 of the chromophore becomes very acid and it transfers its proton to Glu222 by an H-bond chain creating a deprotonated and fluorescent chromophore. This process is called excited-state proton transfer (ESPT). It is completed by backtransfer of the proton in the ground state.
Even in its deprotonated state, the GFP chromophore does not fluoresce forever. At the single molecule level, fluorescence is subjected to temporal fluctuations (blinking) and at some point it ceases irreversibly (bleaching). These processes constitute major annoyances in fluorescence microscopy and are incompletely understood photophysically.
One important activity of our lab is to study photobleaching and photoblinking mechanisms in fluorescent proteins so as to improve their photostability.


Photobleaching is the irreversible light-induced chemical destruction of a fluorophore. In microscopy, loss of fluorescence caused by photobleaching depends on the fluorophore itself and on its nanoenvironment as well as the experimental conditions. Oxygen, temperature, excitation intensity, excitation wavelength, pH, chemical environment of the chromophore can all impact the photobleaching behavior.. We recently found (Duan et al, 2013) that the interplay of available photons and oxygen as a quencher can influence the outcome of the bleaching reaction in the phototransformable fluorescent protein Iris FP.


At the single molecule level, fluorescent proteins blink upon continuous illumination, switching randomly between a fluorescent on-state and a non-fluorescent off-state .
Like photobleaching, blinking mechanisms are also poorly understood. We found that (Adam et al, 2009 ; Roy et al 2011), in IrisFP, a distorted chromophore, potentially due to a coupled electron and proton transfer reaction might be at the origin of blinking.

Optical highlighter fluorescent proteins

A subset of fluorescent proteins -so called phototransformable FPs = PTFPs)- can be manipulated by light to change their fluorescence properties such as emission wavelength or quantum yield.

Photoactivation (irreversible off-on transition) is due to decarboxylation of Glu222 (or the equivalent) followed by conversion of the chromophore from a neutral to anionic state.

Irreversible Green-to-red photoconversion (for Kaede, KikGR, Dendra2, and Eos) occurs when the fluorescent protein is illuminated with ultraviolet or violet radiation (405 nanometers) to induce backbone cleavage between residue 61 and residue 62, leading to subsequent formation of a conjugated dual imidazole ring system.

Reversible Photoswitching (e.g. Dronpa) involves cis-trans photoisomerization induced by alternating radiation between 405 nanometers and 488 nanometers.

IrisFP belongs to two classes, it can be both photoconverted from green to red and in both states, it can be photoswitched between on and off states. It has been our main working horse for mechanistic studies.