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Structural insight into photobleaching mechanisms of reversible photoswitchable fluorescent proteins

Vendredi 5 décembre 2014 14:00 - Duree : 2 heures
Lieu : Salle des séminaires de l’IBS - 71, avenue des Martyrs - Grenoble

Orateur : Soutenance de Thèse de Chanxi DUAN (IBS/DYNAMOP)

The discovery of phototransformable FPs (PTFPs) from Anthozoa species, thanks to their photophysical properties, has opened a large field in biological fluorescence imaging. One of the PTFPs’ sub-groups consists of Reversible Photoswitchable Fluorescent Proteins (RSFPs), which can be reversibly switched between nonfluorescent and fluorescent states. Photobleaching is the permanent loss of the fluorescenceemitting capacity under excitation, which is a common phenomenon among all the fluorescent molecules. Photobleaching has a large impact on the microscopy image quality, notably on super-resolution imaging. Photoswitchable fluorescent proteins have a tendency to lose performance within every switching cycle, a process referred to as “photofatigue”. Our interest of study is focused on the photobleaching mechanisms of RSFPs.

We have reported the crystallographic structure of photobleached IrisFP under high and low illumination intensity at room temperature as well as its spectroscopic modifications. We found that different illumination intensities can result in different photobleaching pathways. Under low illumination intensity, an oxygen-dependent photobleaching pathway was evidenced. Structural modifications induced by singlet-oxygen production within the chromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 and Cys171, locking the chromophore in a nonfluorescent protonated state. Under high illumination intensity, a completely different, oxygen-independent photobleaching pathway was found. The conserved Glu212 underwent decarboxylation concomitantly with an extensive rearrangement of the H-bond network around the chromophore, and an sp2-to-sp3 hybridization change of the carbon atom bridging the chromophore cyclic moieties was observed. As Met159 is the key residue involved in low-intensity illumination photobleaching, we have then mutated Met159 into Alanine in order to avoid sulfoxidation. We found that the IrisFP-M159A mutant display an enhanced photostability in solution, in PVA gel and in E.coli cells.

Contact : ibs.seminaires@ibs.fr

Discipline évènement : (Biologie / Chimie)
Entité organisatrice : (IBS)
Nature évènement : (Soutenance de thèse)
Site de l'évènement : Polygone scientifique

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