Virgile Adam

Virgile Adam
French National Centre for Scientific Research | CNRS · Institut de Biologie Structurale (IBS)

doctor in biophysics

About

57
Publications
11,799
Reads
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1,948
Citations
Introduction
I work at the Institut de Biologie Structurale (IBS), French National Centre for Scientific Research (CNRS). My research focuses on Biochemistry, Biophysics and Biotechnology.
Additional affiliations
October 2011 - present
French National Centre for Scientific Research
Position
  • Researcher
Description
  • Fluorescent proteins; super resolution fluorescence microscopy; TIRF microscopy, single molecule imaging; photoactivation; photobleaching microspectrophotometry ; kinetic crystallography ; structural dynamics ; photophysics
July 2009 - August 2011
KU Leuven
Position
  • PostDoc Position
November 2008 - February 2009
Commissariat à l'Energie Atomique, CEA
Position
  • Research engineer: phototransformable fluorescent proteins and especially their photobleaching process
Education
November 2005 - May 2009

Publications

Publications (57)
Preprint
Full-text available
Single-molecule-localization-microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at 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 the glass transition temperat...
Article
Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy appli...
Preprint
Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applic...
Article
Full-text available
Significance Bacteria possess a sophisticated arsenal of defense mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, Escherichia coli and many other enterobacteria activate a pro...
Article
Green-to-red photoconvertible fluorescent proteins (PCFPs) are key players in advanced microscopy schemes such as photoactivated localization microscopy (PALM). Whereas photoconversion and red-state blinking in PCFPs have been studied intensively, their green-state photophysical behavior has received less attention. Yet dark states in green PCFPs c...
Preprint
Full-text available
Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysi...
Article
Full-text available
Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational cha...
Article
The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essenti...
Article
Full-text available
Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light...
Preprint
Full-text available
The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essenti...
Preprint
Full-text available
Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light...
Article
Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that...
Article
Green-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2 and its derivatives are widely used in PhotoActivated Localization Microscopy (PALM). However, the complex photophysics of these genetically encoded markers complicates the quantitative analysis of PALM data. Here, we show that intense 561-nm light (~1 kW/cm2) typically used t...
Article
Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultra-fast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching fr...
Article
Full-text available
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 fluorescent proteins able to fold and photoswitch in the bacter...
Article
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 the propensity of photoconvertible fluorescent protein markers (PCFPs) to repeatedly enter dark-states. By designi...
Article
Full-text available
Unlabelled: 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 and one allowing elongation. Both cell wall synthesis modes rely on a single cytoskeletal protein, FtsZ. Despite the central role of FtsZ in ovococci,...
Article
Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switc...
Article
In fluorescence microscopy, the photophysical properties of the fluorescent markers play a fundamental role. The beauty of phototransformable fluorescent proteins (PTFPs) is that some of these properties can be precisely controlled by light. A wide range of PTFPs have been developed in recent years, including photoactivatable, photoconvertible and...
Article
Full-text available
Single-molecule localization microscopy of biological samples requires a precise knowledge of the employed fluorescent labels. Photoactivation, photoblinking and photobleaching of phototransformable fluorescent proteins influence the data acquisition and data processing strategies to be used in (Fluorescence) Photoactivation Localization Microscopy...
Article
Full-text available
Fluorescent proteins have revolutionized life sciences because they allow noninvasive and highly specific labeling of biological samples. The subset of "phototransformable" fluorescent proteins recently attracted a widespread interest, as their fluorescence state can be modified upon excitation at defined wavelengths. The fluorescence emission of R...
Article
In these last two decades , fluorescent proteins (FPs) have become highly valued imaging tools for cell biology, owing to their compatibility with living samples, their low levels of invasiveness and the possibility to specifically fuse them to a variety of proteins of interest. Remarkably, the recent development of phototransformable fluorescent p...
Article
Full-text available
The ultrafast excited state dynamics of the fluorescent protein Kaede has been investigated by employing time resolved fluorescence and transient absorption. Upon irradiation of its neutral state, the protein undergoes an efficient conversion to a state that fluoresces at longer wavelengths. The molecular basis of the photoconversion involves an ex...
Article
Photobleaching, the irreversible photo-destruction of a chromophore, severely limits the use of fluorescent proteins (FPs) in optical microscopy. Yet, the mechanisms that govern photobleaching remain poorly understood. In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of FPs that can be repeatedly photoswitched between non-fluorescent...
Article
Green-to-red photoconversion is a reaction that occurs in a limited number of fluorescent proteins and that is currently mechanistically debated. In this contribution we report on our investigation of the photoconvertible fluorescent protein (PCFP) Dendra2 by employing a combination of pump-probe and single photon timing spectroscopic techniques. O...
Article
Phototransformable fluorescent proteins (FPs) have received considerable attention in recent years, because they enable many new exciting modalities in fluorescence microscopy and biotechnology. On illumination with proper actinic light, phototransformable FPs are amenable to long-lived transitions between various fluorescent or nonfluorescent stat...
Article
Full-text available
Proteins of the GFP (green fluorescent protein) family have revolutionized life sciences because they allow the tagging of biological samples in a non-invasive genetically encoded way. 'Phototransformable' fluorescent proteins, in particular, have recently attracted widespread interest, as their fluorescence state can be finely tuned by actinic lig...
Article
Fluorescent proteins (FPs) of the green fluorescent protein family blink and bleach like all fluorophores. However, contrary to organic dyes, the mechanisms by which transient losses of fluorescence occur in FPs have received little attention. Here, we focus on the photoactivatable IrisFP, for which a transient non-fluorescent chromophoric state wi...
Article
Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent...
Article
Combining kinetic crystallography and optical spectroscopy for studying fluorescent proteins Every year, the three-dimensional structures of numerous and increasingly complex biological macromolecules are obtained by X-ray crystallography, using synchrotron radiation. However, the knowledge of the spatial organization of a protein in the resting st...
Article
Fluorescent proteins from the GFP family have become indispensable imaging tools in life sciences research. In recent years, a wide variety of these proteins were discovered in non-bioluminescent anthozoa. Some of them feature exciting new properties, including the possibility to change their fluorescence quantum yield and/or color by irradiating w...
Article
The recent discovery of photoconvertible and photoswitchable fluorescent proteins (PCFPs and RSFPs, respectively) that can undergo photoinduced changes of their absorption/emission spectra opened new research possibilities in subdiffraction microscopy and optical data storage. Here we demonstrate the proof-of-principle for read only and rewritable...
Article
Full-text available
We have studied the photoswitching behaviour of a number of photochromic fluorescent proteins at cryo-temperature. Spectroscopic investigations at the ensemble level showed that EYFP, Dronpa and IrisFP all exhibit reversible photoswitching at 100 K, albeit with a low quantum yield. The photophysics of the process were studied in more details in the...
Article
Dendra2 is an engineered, monomeric GFP-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green- to a red-emitting state upon exposure to purple-blue light. We have measured the X-ray structure of the green species of Dendra2 and performed a comprehensive characterization of the optical...
Article
We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumin...
Article
Fluorescent proteins undergoing green to red photoconversion have proved to be essential tools in cell biology, notably in superlocalization nanoscopy. However, the exact mechanism governing photoconversion, which overall involves irreversible cleavage of the protein backbone and elongation of the chromophore pi-conjugation, remains unclear. In thi...
Article
Full-text available
Mechanistic investigations of biological enzymatic processes require controlled initiation and monitoring of catalytic reactions. A well-known technique to trap and observe reaction intermediates building up along a reaction pathway is the use of low temperature conditions. Here, we report a kinetically competent system for the release of molecular...
Thesis
Full-text available
Since the discovery of the green fluorescent protein (GFP) in 1962, many developments allowed improving the use of this naturally light-emitting protein as a powerful tool for tracking proteins or organelles of interest within living cells and organisms. At the beginning of the 21st century, the discovery of photoactivatable fluorescent proteins (P...
Article
Dendra2 is an engineered, monomeric GFP-like protein that belongs to a subclass of fluorescent proteins undergoing irreversible photoconversion from a green- to a red-emitting state upon exposure to purple-blue light. This photoinduced process occurs only in the neutral state of the chromophore and is known to result from backbone cleavage accompan...
Article
Full-text available
Photoactivatable fluorescent proteins (FPs) are powerful fluorescent highlighters in live cell imaging and offer perspectives for optical nanoscopy and the development of biophotonic devices. Two types of photoactivation are currently being distinguished, reversible photoswitching between fluorescent and nonfluorescent forms and irreversible photoc...
Article
Full-text available
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 of the structure of a protein or to monitor changes that may occur during X-ray diffraction data collection. Microspectrophotometers in the UV-visible abso...
Article
Full-text available
Iron-peroxide intermediates are central in the reaction cycle of many iron-containing biomolecules. We trapped iron(III)-(hydro)peroxo species in crystals of superoxide reductase (SOR), a nonheme mononuclear iron enzyme that scavenges superoxide radicals. X-ray diffraction data at 1.95 angstrom resolution and Raman spectra recorded in crystallo rev...
Article
Full-text available
The superoxide radical O2·̅ is a toxic by-product of oxygen metabolism. Two O2·̅ detoxifying enzymes have been described so far, superoxide dismutase and superoxide reductase (SOR), both forming H2O2 as a reaction product. Recently, the SOR active site, a ferrous iron in a [Fe²⁺ (N-His)4 (S-Cys)] pentacoordination, was shown to have the ability to...
Article
Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis thymidylate kinase (TMPK(Mtub)) catalyzes the ATP-dependent phosphorylation of deoxythymidine 5'-monophosphate (dTMP). Essential to DNA replication, this enzyme represents a promising target for developing new drugs against TB, because the conf...
Article
Full-text available
Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O2*-). SOR catalyses the one-electron reduction of O2*- to hydrogen peroxide at a nonheme ferrous iron center. The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocya...
Article
Full-text available
Absorption microspectrophotometry has been shown to be of considerable help to probe crystalline proteins containing chromophores, metal centres, or coloured substrates/co-factors. Absorption spectra contribute to the proper interpretation of crystallographic structures, especially when transient intermediate states are studied. Here it is shown th...
Article
Depuis la découverte de la protéine fluorescente verte (GFP) en 1962, de nombreux développements ont permis d'améliorer l'utilisation de cette protéine naturellement luminescente en tant que puissant outil permettant de suivre des protéines ou des organelles d'intérêt dans les cellules ou organismes vivants. Au début du 21ème siècle, la découverte...

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Projects

Projects (2)
Archived project
Slowing down enzyme reactions and/or making X-ray probing faster/gentler in order to capture transient catalytic intermediates