A5020225059- Advanced Fluorescence Microscopy Techniques
- Photoreceptor and optogenetics research
- Photosynthetic Processes and Mechanisms
- Photochromic and Fluorescence Chemistry
- Advanced Electron Microscopy Techniques and Applications
- Cell Image Analysis Techniques
- Metal-Catalyzed Oxygenation Mechanisms
- bioluminescence and chemiluminescence research
- Enzyme Structure and Function
- Porphyrin and Phthalocyanine Chemistry
- Click Chemistry and Applications
- Metal complexes synthesis and properties
- Tuberculosis Research and Epidemiology
- Semiconductor materials and devices
- Computational Drug Discovery Methods
- Laser-Matter Interactions and Applications
- Photodynamic Therapy Research Studies
- Biochemical and Molecular Research
- Advanced NMR Techniques and Applications
- Luminescence Properties of Advanced Materials
- Spectroscopy Techniques in Biomedical and Chemical Research
- Mass Spectrometry Techniques and Applications
- Electron Spin Resonance Studies
- Advanced Biosensing Techniques and Applications
- Traditional Chinese Medicine Analysis
Institut de Biologie Structurale
2015-2024
Commissariat à l'Énergie Atomique et aux Énergies Alternatives
2007-2023
Université Grenoble Alpes
2012-2023
CEA Grenoble
2007-2023
Centre National de la Recherche Scientifique
2012-2023
Laboratoire de Tribologie et Dynamique des Systèmes
2020
University of Southampton
2016
European Synchrotron Radiation Facility
2006-2015
Institut de Recherches en Technologies et Sciences pour le Vivant
2007-2013
Laboratoire Physiologie Cellulaire & Végétale
2013
Photoactivatable fluorescent proteins (FPs) are powerful highlighters in live cell imaging and offer perspectives for optical nanoscopy the development of biophotonic devices. Two types photoactivation currently being distinguished, reversible photoswitching between nonfluorescent forms irreversible photoconversion. Here, we have combined crystallography (in crystallo) spectroscopy to characterize Phe-173-Ser mutant tetrameric variant EosFP, named IrisFP, which incorporates both...
Iron-peroxide intermediates are central in the reaction cycle of many iron-containing biomolecules. We trapped iron(III)-(hydro)peroxo species crystals superoxide reductase (SOR), a nonheme mononuclear iron enzyme that scavenges radicals. X-ray diffraction data at 1.95 angstrom resolution and Raman spectra recorded crystallo revealed iron-(hydro)peroxo with (hydro)peroxo group bound end-on. The dynamic SOR active site promotes formation transient hydrogen bond networks, which presumably...
Abstract Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off - state to on involves trans -to- cis chromophore isomerization and proton transfer. Whereas excited-state events the ps timescale have been structurally characterized, conformational changes slower timescales remain elusive. Here we describe off-to-on photoswitching mechanism RSFP rsEGFP2 by using combination of time-resolved serial...
Dendra2 is an engineered, monomeric GFP-like protein that belongs to a subclass of fluorescent proteins undergoing irreversible photoconversion from green- red-emitting state upon exposure purple-blue light. This photoinduced process occurs only in the neutral chromophore and known result backbone cleavage accompanied by extension delocalized π-electron system. We have measured X-ray structure green species performed comprehensive characterization optical absorption fluorescence properties...
Photobleaching, the irreversible photodestruction of a chromophore, severely limits use fluorescent proteins (FPs) in optical microscopy. Yet, mechanisms that govern photobleaching remain poorly understood. In Reversibly Switchable Fluorescent Proteins (RSFPs), class FPs can be repeatedly photoswitched between nonfluorescent and states, achievable number switching cycles, process known as photofatigue. We investigated photofatigue protein IrisFP using combined X-ray crystallography,...
Ovococci form a morphological group that includes several human pathogens (enterococci and streptococci). Their shape results from two modes of cell wall insertion, one allowing division elongation. Both synthesis rely on single cytoskeletal protein, FtsZ. Despite the central role FtsZ in ovococci, detailed view vivo nanostructure ovococcal Z-rings has been lacking thus far, limiting our understanding their assembly architecture. We have developed use photoactivated localization microscopy...
Abstract Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable able fold and photoswitch bacterial periplasm, rsFolder rsFolder2. was designed by hybridisation Superfolder-GFP with rsEGFP2 inherited fast folding properties former together rapid switching latter,...
Photoactivated localization microscopy (PALM) is a powerful technique to investigate cellular nanostructures quantitatively and dynamically. However, the use of PALM for molecular counting or single-particle tracking remains limited by propensity photoconvertible fluorescent protein markers (PCFPs) repeatedly enter dark states. By designing single mutants mEos2-A69T Dendra2-T69A, we completely swapped blinking behaviors mEos2 Dendra2, two popular PCFPs. We combined X-ray crystallography...
Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump–probe absorption spectroscopy reversibly protein IrisFP solution to study photoswitching from nonfluorescent (off) (on) state. Evidence is provided for existence of several intermediate states pico- and microsecond scales that are attributed chromophore isomerization proton...
Green-to-red photoconvertible fluorescent proteins (PCFPs) are key players in advanced microscopy schemes such as photoactivated localization (PALM). Whereas photoconversion and red-state blinking PCFPs have been studied intensively, their green-state photophysical behavior has received less attention. Yet dark states green can become strongly populated PALM exert an indirect but considerable influence on the quality of data recorded red channel. Furthermore, photoswitching be used directly...
We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This is assigned to radical species that either relaxes ground or evolves into permanently bleached chromophore. took advantage of X-rays populate radical, which presumably forms under illumination visible light by an electron-transfer reaction triplet state. The combined X-ray diffraction and crystallo UV−vis absorption, fluorescence, Raman data reveal formation involves...
Absorption microspectrophotometry has been shown to be of considerable help probe crystalline proteins containing chromophores, metal centres, or coloured substrates/co-factors. spectra contribute the proper interpretation crystallographic structures, especially when transient intermediate states are studied. Here it is that fluorescence might also used for such purposes if endogenous fluorophores present in macromolecule exogenous added and either bind protein reside solvent channels. An...
Synchrotrons are now producing thousands of macromolecular structures each year. The need for complementary techniques available on site has progressively emerged, either to assess the relevance structure a protein or monitor changes that may occur during X-ray diffraction data collection. Microspectrophotometers in UV–visible absorbance fluorescence mode have evolved over past few decades become instruments choice perform such tests. Described here recent improvements microspectrophotometer...
Fluorescent proteins (FPs) of the green fluorescent protein family blink and bleach like all fluorophores. However, contrary to organic dyes, mechanisms by which transient losses fluorescence occur in FPs have received little attention. Here, we focus on photoactivatable IrisFP, for a non-fluorescent chromophoric state with distorted geometry was recently reported (Adam, V.; et al. J. Am. Chem. Soc. 009, 131, 18063). We investigated chemical nature this blinked employing quantum...
Single-molecule localization microscopy of biological samples requires a precise knowledge the employed fluorescent labels. Photoactivation, photoblinking and photobleaching phototransformable proteins influence data acquisition processing strategies to be used in (Fluorescence) Photoactivation Localization Microscopy ((F)-PALM), notably for reliable molecular counting. As these parameters might depend on local environment, they should measured cellulo biologically relevant experimental...
Single-molecule localization microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below glass-transition hampers efficient cryo-photoswitching. We investigated cryo-switching rsEGFP2, one most reversibly switchable ambient due facile cis–trans isomerization...