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Lighting up proteins in living systems
By Arnaud Gautier (ENS, Dept. de Chimie, Paris)
June 29, 2015 at 11:00AM - Salle de séminaires 5ème étagé, Tour 32-33



Deciphering the complex mechanisms controlling cells and organisms requires effective imaging systems and fluorescent probes to observe biomolecules in real time with high spatiotemporal resolution. Ideally, fluorescent probes should be highly specific for their target, bright and photostable, non-toxic, and the smallest as possible to avoid perturbing the function of their target. They should also exhibit instantaneous and robust fluorescence, and offer the possibility to tune at will the fluorescence of the system for sophisticated imaging protocols. During this seminar, I will present the development of Y-FAST, a tunable protein tag, monomeric and twice smaller than the green fluorescent protein (GFP), whose yellow fluorescence can be turned on and off at will by addition or withdrawal of a non-toxic fluorogenic ligand. Upon binding, the fluorogen undergoes not only fluorescence activation but also specific absorption red shift. This latter spectral modification enables to distinguish free and bound fluorogen via the choice of the excitation wavelength, increasing thus specificity. This Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), evolved from the photoactive yellow protein (PYP), is as bright as common fluorescent proteins, exhibits good photostability and allows the efficient labeling of proteins in various hosts from microorganisms to mammalian cells and zebrafish embryos. Furthermore, the rapid exchange dynamics of the system enables to rapidly label and unlabel proteins by addition and withdrawal of the fluorogen, opening new possibilities for multiplexing experiments.