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Towards a Better Understanding of Photobleaching in Fluorescent Proteins

Vendredi 17 janvier 2014 11:00 - Duree : 1 heure
Lieu : Salle des séminaires de l’IBS - 6 rue Jules Horowitz - Grenoble

Orateur : Chenxi DUAN (Institut de Biologie Structurale Groupe Dynamique et Cinétique des processus moléculaires)

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. Large efforts have been dedicated to the development of derivatives from GFP and fluorescent proteins from other marine species. So-called phototransformable fluorescent proteins (PTFPS) have been engineered and show a lot of interesting photophysical properties. PTFPs are largely used in super-resolution fluorescence microscopies and many other advanced imaging techniques as well as biotechnology applications. The subset of reversibly switchable fluorescent proteins (RSFPs) can be repeatedly switched between non-fluorescent and fluorescent states upon illumination with light under proper wavelengths. Photobleaching, the permanent loss of fluorescence emission, is a general phenomenon amongst all existing fluorophores. In RSFPs, photobleaching manifests itself in a process referred to as “photofatigue”. Photofatigue events that eventually occur after several on-off switching cycles in RSFPs is one crucial limiting parameter of the imaging techniques mentioned above. IrisFP is a fascinating RSFP that has been thoroughly studied at IBS in the Pixel team since several years. During my PhD work, we investigated photofatigue mechanisms in IrisFP by combining kinetic X-ray crystallography, optical in crystallo spectroscopy, mass spectrometry, super-resolution microscopy and modeling approaches. We identified two completely different mechanisms only depending on the intensity of the used illumination. Our findings could possibly be related to the phototoxic effect in living cells during fluorescence microscopy imaging. We will present in detail the structural changes and chemical modifications of the IrisFP chromophore and its micro-environment in these two photofatigue mechanisms as well as the improvement of photostability that could be obtained by rationally designing specific mutants.

Attention !! Les séminaires ont désormais lieu dans notre nouveau bâtiment sur EPN Campus. L’accès au campus EPN nécessite un avis de rendez-vous. Contactez ibs.seminaires@ibs.fr

Contact : odile.kaikati@ibs.fr

Discipline évènement : (Biologie / Chimie)
Entité organisatrice : (IBS)
Nature évènement : (Séminaire)
Evènement répétitif : (Séminaire IBS)
Site de l'évènement : Polygone scientifique

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