Laboratoire P.A.S.T.E.U.R

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Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration

J. Phys. Chem. Lett. 2017, 8, 1489−1495


Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the fi rst timeresolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the picosecond− nanosecond regime that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule.




Selective Electrochemical Bleaching of the Outer Leaflet of Fluorescently Labeled Giant Liposomes

Chem. Eur. J., 23,1–8, 2017


Electrochemistry and confocal fluorescence microscopy were successfully combined to selectively bleach and monitor the fluorescence of NBD (7-nitrobenz-2-oxa-1,3- diazole)-labeled phospholipids of giant liposomes. Three types of giant unilamellar vesicles have been investigated, the fluorescent phospholipids being localized either mainly on their outer-, inner-, or both inner/outer leaflets. We established that only the fluorescent lipids incorporated in the outer leaflet of the vesicles underwent electrochemical bleaching upon reduction. The relative fluorescence intensity decay was quantified all along the electrochemical extinction through an original fluorescence loss in electrobleaching (FLIE) assay. As expected, the reorganization of the fluorescent phospholipids followed diffusion-driven dynamics. This was also evidenced by comparison with fluorescence loss in photobleaching (FLIP) and the corresponding numerical model. The value of the lateral diffusion coefficient of phospholipids was found to be similar to that obtained by other methods reported in the literature. This versatile and selective bleaching procedure appears reliable to explore important biological and pharmacological issues.


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A Dual Functional Electroactive and Fluorescent Probe for Coupled Measurements of Vesicular Exocytosis with High Spatial and Temporal Resolution

Angew. Chem. Int. Ed. 2017, 56, 1 – 6


In this work, Fluorescent False Neurotransmitter 102 (FFN102), a synthesized analogue of biogenic neurotransmitters, was demonstrated to show both pH-dependent fluorescence and electroactivity. To study secretory behaviors at the single-vesicle level, FFN102 was employed as a new fluorescent/electroactive dual probe in a coupled technique (amperometry and total internal reflection fluorescence microscopy (TIRFM)). We used N13 cells, a stable clone of BON cells, to specifically accumulate FFN102 into their secretory vesicles, and then optical and electrochemical measurements of vesicular exocytosis were experimentally achieved by using indium tin oxide (ITO) transparent electrodes. Upon stimulation, FFN102 started to diffuse out from the acidic intravesicular microenvironment to the neutral extracellular space, leading to fluorescent emissions and to the electrochemical oxidation signals that were simultaneously collected from the ITO electrode surface. The correlation of fluorescence and amperometric signals resulting from the FFN102 probe allows realtime monitoring of single exocytotic events with both high spatial and temporal resolution. This work opens new possibilities in the investigation of exocytotic mechanisms.