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PhD thesis :  Mechanistic studies of photoactivatable fluorescent proteins: a combined approach by crystallography and spectroscopy

Thesis of Adam Virgile



 Since the discovery of the green fluorescent protein (GFP) in 1962, many developments allowed improving the use of this naturally light-emitting protein as a powerful tool for tracking proteins or organelles of interest within living cells and organisms. At the beginning of the 21st century, the discovery of photoactivatable fluorescent proteins (PAFPs), notably from Anthozoan species, triggered a revolution in the field of FP technology.


Some PAFPs are capable of being irreversibly photoconverted from a green- to a red-emitting form while other ones can be reversibly switched on and off, depending on specific excitation wavelengths. These proteins are being extensively used in optical microscopy techniques, particularly in “nanoscopy”, which provides optical resolution 10 fold beyond the theoretical Abbe limit. In order to further develop these techniques, notably in term of time-resolution, the need to obtain brighter fluorescent probes that photoconvert or photoswitch efficiently is crucial.


At the same time, fluorescent highlighters generally need to be monomeric and photostable. In order to better understand the mechanisms of phototransformations in PAFPs, three members of the family have been studied: EosFP, Dendra2 and IrisFP. The phenomena of green-to-red photoconversion, reversible photoswitching and non-reversible photobleaching have been studied by a combination of X-ray crystallography and microspectrophotometry using the Cryobench laboratory of the ESRF/IBS.


Together, the results have allowed us to propose a mechanism for the photoconversion of EosFP and Dendra2 and to discover and characterize IrisFP, the first PAFP combining both properties of photoconversion and photoswitching. The structural modifications of the chromophore associated with an X-ray induced radical state, likely to be involved in the photobleaching pathway of PAFPs, were also characterized.


keywords: Fluorescent proteins – photoconversion – photoswitching – photobleaching – protein crystallography – microspectrophotometry – synchrotron – Cryobench

Because kinetic crystallography is, by essence, interested in initiating and following structural modifications that occur along a reaction (such as trapping reaction intermediate states), we often used lasers combined to X-rays to trig reactions within protein crystals.

Doing so, we were interested in photosensitive compounds and proteins and it appeared natural to focus our efforts toward newly discovered phototransformable fluorescent proteins (PTFPs).

PTFPs are macromolecules that comprise a genetically-encoded fluorophore. The best known member is the green fluorescent protein (GFP) whose discoverers and developers have been later awarded the Nobel Prize in Chemistry 2008

Unlike GFP, PTFPs are also capable of irreversible and/or reversible photo-induced transformations that give them the ability to either be activated to a glowing form or to reversibly extinct their fluorescence and even to emit light in another color.


These fascinating transformations were badly understood at the time of my PhD thesis and we decided to study their mechanisms by kinetic crystallography. I was lucky enough to make my PhD thesis at the ESRF, within the macromolecular crystallography group under the supervision of Dr. Dominique Bourgeois and Dr. Sean McSweeney.

In summary, during this PhD thesis, we better understood the structural mechanisms of reversible fluorescence switching and irreversible colour photoconversion and discovered IrisFP, first member of a family combining both properties of irreversible photoconversion and reversible photoswitching.

PhD diploma of Adam Virgile

Left photo = During the defense in the ESRF auditorium, Middle photo = Virgile Adam, Dominique Bourgeois and Daniel Picot, Right photo = PhD diploma


Left photo = From left to right: S. McSweeney, G.U. Nienhaus, D. Bourgeois, V. Adam, D. Picot, J. Hofkens, M. Robert-Nicoud and C. Royer

Right photo = ESRF-synchrotron-shaped cake during the thesis party

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