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Études structurales de la dynamique de protéines fluorescentes vertes et jaunes utilisées en imagerie cellulaire

Abstract : Fluorescent Proteins (FPs) homologous to AvGFP (Green Fluorescent Protein from the jellyfish Aequoria victoria) are versatile tools used in live cell imaging. The amount of information that can be derived from the fluorescence signals depends on the spectroscopic performances of the FP. The development of new FPs should focus on both brightness increase and control of the protein response to physicochemical parameter variations within the cell. Current yellow FPs exhibit a strong sensitivity to pH. In order to engineer less sensitive variants, two complementary approaches have been used: the first one consists in studying the influence of the hydrogen bond network dynamics around the chromophore on its protonation state. In the second one, I have sought to identify the structural determinants of the particularly high brightness of newly discovered yellow FPs from a sea worm, Branchiostoma lanceolatum.First, I wrote an algorithm that can identify all hydrogen bonds within a protein and analyse their dynamics along molecular dynamics simulations. It allows for their clustering in networks, the identification of networks connected to a given atom and the monitoring of their dynamics. The method was validated by using the algorithm on various AvGFP mutants for which excited state proton transfer has been experimentally studied. This algorithm should also be useful for the study of other biological systems whose function is based on proton transfer.Besides, I solved the structure of the natural yellow FP lanYFP from Branchiostoma lanceolatum, which is particularly bright, but presents a tetrameric arrangement. This protein was monomerized by directed evolution, which led to the yellow-green FP mNeonGreen, now a benchmark in this spectral range. I also solved the structure of mNeonGreen, which allowed me to rationalize a posteriori the mutations that have been introduced during the evolution process. Finally, I performed a specific radiation damage study in order to explain the remarkable change in colour of mNeonGreen crystals upon X-ray data collection. Altogether, the results of my PhD work provides a theoretical and experimental framework of the determinants that drive the fluorescence properties of yellow FPs.
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Damien Clavel. Études structurales de la dynamique de protéines fluorescentes vertes et jaunes utilisées en imagerie cellulaire. Cristallographie. Université Paris Saclay (COmUE), 2016. Français. ⟨NNT : 2016SACLS491⟩. ⟨tel-01466736⟩

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