Dominique Bourgeois

French National Centre for Scientific Research, Lutetia Parisorum, Île-de-France, France

Are you Dominique Bourgeois?

Claim your profile

Publications (106)458.83 Total impact

  • Source
    [Show abstract] [Hide abstract]
    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 fluorescent proteins able to fold and photoswitch in the bacterial periplasm, rsFolder and rsFolder2. rsFolder was designed by hybridisation of Superfolder-GFP with rsEGFP2, and inherited the fast folding properties of the former together with the rapid switching of the latter, but at the cost of a reduced switching contrast. Structural characterisation of the switching mechanisms of rsFolder and rsEGFP2 revealed different scenarios for chromophore cis-trans isomerisation and allowed designing rsFolder2, a variant of rsFolder that exhibits improved switching contrast and is amenable to RESOLFT nanoscopy. The rsFolders can be efficiently expressed in the E. coli periplasm, opening the door to the nanoscale investigation of proteins localised in hitherto non-observable cellular compartments.
    Full-text · Article · Jan 2016 · Scientific Reports
  • Romain Berardozzi · Virgile Adam · Alexandre Martins · Dominique Bourgeois
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 designing the single-mutants mEos2-A69T and Dendra2-T69A, we completely swapped the blinking behaviors of mEos2 and Dendra2, two popular PCFPs. We combined X-ray crystallography and single-molecule microscopy to show that blinking in mEos2 and Dendra2 is largely controlled by the orientation of arginine 66, a highly-conserved residue in Anthozoan PCFPs. The Arg66 side-chain conformation affects the bleaching and the on-to-off transition quantum yields, as well as the fraction of molecules entering long-lived dark states, resulting in widely different apparent blinking behaviors that largely modulate the efficiency of current blinking correction procedures. The present work provides mechanistic insight into the complex photophysics of Anthozoan PCFPs and will facilitate future engineering of bright and low-blinking variants suitable for PALM microscopy.
    No preview · Article · Dec 2015 · Journal of the American Chemical Society
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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, a detailed view of the in vivo nanostructure of ovococcal Z-rings has been lacking thus far, limiting our understanding of their assembly and architecture. We have developed the use of photoactivated localization microscopy (PALM) in the ovococcus human pathogen Streptococcus pneumoniae by engineering spDendra2, a photoconvertible fluorescent protein optimized for this bacterium. Labeling of endogenously expressed FtsZ with spDendra2 revealed the remodeling of the Z-ring’s morphology during the division cycle at the nanoscale level. We show that changes in the ring’s axial thickness and in the clustering propensity of FtsZ correlate with the advancement of the cell cycle. In addition, we observe double-ring substructures suggestive of short-lived intermediates that may form upon initiation of septal cell wall synthesis. These data are integrated into a model describing the architecture and the remodeling of the Z-ring during the cell cycle of ovococci.
    Full-text · Article · Aug 2015 · mBio
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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 switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions.
    No preview · Article · Feb 2015
  • Virgile Adam · Dominique Bourgeois

    No preview · Article · Jan 2015
  • Virgile Adam · Romain Berardozzi · Martin Byrdin · Dominique Bourgeois
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 photoswitchable fluorescent proteins. These smart labels triggered a plethora of advanced fluorescence methods to scrutinize biological cells or organisms dynamically, quantitatively and with unprecedented resolution. Despite continuous improvements, PTFPs still suffer from limitations, and mechanistic questions remain as to how these proteins precisely work.
    No preview · Article · Jun 2014 · Current Opinion in Chemical Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 ((F)-PALM), notably for reliable molecular counting. As these parameters might depend on the local environment, they should be measured in cellulo in biologically relevant experimental conditions. Here, we measured phototransformation quantum yields for Dendra2 fused to actin in fixed mammalian cells in typical (F)-PALM experiments. To this aim, we developed a data processing strategy based on the clustering optimization procedure proposed by Lee et al (PNAS 109, 17436–17441, 2012). Using simulations, we estimated the range of experimental parameters (molecular density, molecular orientation, background level, laser power, frametime) adequate for an accurate determination of the phototransformation yields. Under illumination at 561 nm in PBS buffer at pH 7.4, the photobleaching yield of Dendra2 fused to actin was measured to be (2.5±0.4)×10−5, whereas the blinking-off yield and thermally-activated blinking-on rate were measured to be (2.3±0.2)×10−5 and 11.7±0.5 s−1, respectively. These phototransformation yields differed from those measured in poly-vinyl alcohol (PVA) and were strongly affected by addition of the antifading agent 1,4-diazabicyclo[2.2.2]octane (DABCO). In the presence of DABCO, the photobleaching yield was reduced 2-fold, the blinking-off yield was decreased more than 3-fold, and the blinking-on rate was increased 2-fold. Therefore, DABCO largely improved Dendra2 photostability in fixed mammalian cells. These findings are consistent with redox-based bleaching and blinking mechanisms under (F)-PALM experimental conditions. Finally, the green-to-red photoconversion quantum yield of Dendra2 was estimated to be (1.4±0.6)×10−5 in cellulo under 405 nm illumination.
    Full-text · Article · Jun 2014 · PLoS ONE
  • Source
    Chenxi Duan · Virgile Adam · Martin Byrdin · Dominique Bourgeois
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 Reversibly Switchable Fluorescent Proteins (RSFPs), in particular, can be repeatedly switched on and off. RSFPs enable many new exciting modalities in fluorescence microscopy and biotechnology, including protein tracking, photochromic Förster Resonance Energy Transfer, super-resolution microscopy, optogenetics, and ultra-high-density optical data storage. Photoswitching in RSFPs typically results from chromophore cis-trans isomerization accompanied by a protonation change, but other switching schemes based on, e.g., chromophore hydration/dehydration have also been discovered. In this chapter, we review the main structural features at the basis of photoswitching in RSFPs.
    Full-text · Article · Apr 2014 · Methods in molecular biology (Clifton, N.J.)
  • Chenxi Duan · Virgile Adam · Martin Byrdin · Dominique Bourgeois

    No preview · Article · Mar 2014
  • Virgile Adam · Romain Berardozzi · Martin Byrdin · Dominique Bourgeois
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 photoswitchable fluorescent proteins. These smart labels triggered a plethora of advanced fluorescence methods to scrutinize biological cells or organisms dynamically, quantitatively and with unprecedented resolution. Despite continuous improvements, PTFPs still suffer from limitations, and mechanistic questions remain as to how these proteins precisely work.
    No preview · Article · Jan 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 and fluorescent states, photobleaching limits the achievable number of switching cycles, a process known as photofatigue. We investigated the photofatigue mechanisms in the protein IrisFP using combined X-ray crystallography, optical in crystallo spectroscopy, mass spectrometry and modeling approaches. At laser-light intensities typical of conventional wide-field fluorescence microscopy, an oxygen-dependent photobleaching pathway was evidenced. Structural modifications induced by singlet-oxygen production within the chromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 and Cys171, locking the chromophore in a non-fluorescent protonated state. At laser-light intensities typical of localization-based nanoscopy (>0.1 kW/cm²), a completely different, oxygen-independent photobleaching pathway was found to take place. The conserved Glu212 underwent decarboxylation concomitantly with an extensive rearrangement of the H-bond network around the chromophore, and a sp2-to-sp3 hybridization change of the carbon atom bridging the chromophore cyclic moieties was observed. This two-regime photobleaching mechanism is likely to be a common feature in RSFPs from Anthozoan species, which typically share high structural and sequence identity with IrisFP. In addition, our results suggest that, when such FPs are used, the illumination conditions employed in localization-based super-resolution microscopy might generate less cytotoxicity than those of standard wide-field microscopy, at constant absorbed light-dose. Finally, our data will facilitate the rational design of FPs displaying enhanced photo-resistance.
    Full-text · Article · Sep 2013 · Journal of the American Chemical Society

  • No preview · Article · Aug 2012
  • Source
    Dominique Bourgeois · Virgile Adam
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 states, resulting in processes known as photoactivation, photoconversion, or photoswitching. Here, we review the subclass of photoswitchable FPs with a mechanistic perspective. These proteins offer the widest range of practical applications, including reversible high-density data bio-storage, photochromic FRET, and super-resolution microscopy by either point-scanning, structured illumination, or single molecule-based wide-field approaches. Photoswitching can be engineered to occur with high contrast in both Hydrozoan and Anthozoan FPs and typically results from a combination of chromophore cis-trans isomerization and protonation change. However, other switching schemes based on, for example, chromophore hydration/dehydration have been discovered, and it seems clear that ever more performant variants will be developed in the future.
    Full-text · Article · Jun 2012 · International Union of Biochemistry and Molecular Biology Life
  • Source
    Dominique Bourgeois · Aline Regis-Faro · Virgile Adam
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 light, a property central to the development of super-resolution microscopy. Beyond microscopy applications, phototransformable fluorescent proteins are also exquisite tools to investigate fundamental protein dynamics. Using light to trigger processes such as photoactivation, photoconversion, photoswitching, blinking and photobleaching allows the exploration of the conformational landscape in multiple directions. In the present paper, we review how structural dynamics of phototransformable fluorescent proteins can be monitored by combining X-ray crystallography, in crystallo optical spectroscopy and simulation tools such as quantum chemistry/molecular mechanics hybrid approaches. Besides their usefulness to rationally engineer better performing fluorescent proteins for nanoscopy and other biotechnological applications, these investigations provide fundamental insights into protein dynamics.
    Full-text · Article · Jun 2012 · Biochemical Society Transactions
  • Arijit Roy · Martin J Field · Virgile Adam · Dominique Bourgeois
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 with distorted geometry was recently reported (Adam, V.; et al. J. Am. Chem. Soc. 009, 131, 18063). We investigated the chemical nature of this blinked state by employing quantum chemical/molecular mechanical calculations. Our findings suggest two previously unidentified dark states that display similar distorted chromophores with a transiently ruptured π-electron system. Both are protonated at atom C(α) of the chromophore methylene bridge. Transient protonation may occur via proton transfer from the nearby Arg66 either in the triplet state T(1) after intersystem crossing or in an anionic radical (doublet) ground state. As Arg66 is conserved in green-to-red photoconvertible FPs, these dark states are predicted to be common to all these proteins. We also suggest that C(α) protonated dark states may accelerate photobleaching by favoring decarboxylation of the fully conserved Glu212.
    No preview · Article · Nov 2011 · Journal of the American Chemical Society
  • D. Arcizet · V. Adam · P. Carpentier · A. Faro · D. Bourgeois
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 state is often insufficient to understand its biological function. Indeed, protein function results from the conformational changes that take place during the reaction cycle. In order to understand this structure-dynamics-function relationship, it is fundamental to resort to .kinetic. crystallography, a technique allowing the determination of protein structures in different states along their reaction cycle. Moreover, the characterization of these various states by optical spectroscopy is often crucial to correctly identify them, and to gather complementary knowledge. This article describes how fruitful the coupling between kinetic crystallography and optical spectroscopy can be for the study of fluorescent proteins of the GFP family, with a focus on phototransformable fluorescent proteins, whose complex photophysical properties can be understood through the correlation, for each fluorescent state, between the structure (obtained by X-ray crystallography) and the spectroscopic signature of the electronic and vibrational states (respectively measured by absorption/fluorescence and Raman spectroscopy). The fascinating case of the protein IrisFP is detailed.
    No preview · Article · Oct 2011
  • [Show abstract] [Hide abstract]
    ABSTRACT: Photoactivatable fluorescent proteins are essential players in nanoscopy approaches based on the super-localization of single molecules. The subclass of reversibly photoswitchable fluorescent proteins typically activate through isomerization of the chromophore coupled with a change in its protonation state. However, the interplay between these two events, the details of photoswitching pathways, and the role of protein dynamics remain incompletely understood. Here, by using a combination of structural and spectroscopic approaches, we discovered two fluorescent intermediate states along the on-switching pathway of the fluorescent protein Padron. The first intermediate can be populated at temperatures as low as 100 K and results from a remarkable trans-cis isomerization of the anionic chromophore taking place within a protein matrix essentially deprived of conformational flexibility. This intermediate evolves in the dark at cryotemperatures to a second structurally similar but spectroscopically distinct anionic intermediate. The final fluorescent state, which consists of a mixture of anionic and neutral chromophores in the cis configuration, is only reached above the glass transition temperature, suggesting that chromophore protonation involves solvent interactions mediated by pronounced dynamical breathing of the protein scaffold. The possibility of efficiently and reversibly photoactivating Padron at cryotemperatures will facilitate the development of advanced super-resolution imaging modalities such as cryonanoscopy.
    No preview · Article · Sep 2011 · Journal of the American Chemical Society
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 with light of specific wavelengths. These photoactivatable fluorescent proteins enable many interesting applications including pulse-chase experiments and super-resolution imaging. In this review, we discuss the development of advanced variants, using a structure-function based, molecular biophysics approach, of the photoactivatable fluorescent protein EosFP, which can be photoconverted from green to red fluorescence by ~400 nm light. A variety of applications are presented that demonstrate the versatility of these marker proteins in live-cell imaging.
    Full-text · Article · Jun 2011 · Journal of Biophotonics
  • Source
    John E McGeehan · Dominique Bourgeois · Antoine Royant · Philippe Carpentier
    [Show abstract] [Hide abstract]
    ABSTRACT: Raman spectroscopy is a powerful technique that, in recent years, has been successfully coupled to X-ray crystallography for the analysis of biological macromolecular systems. The complementarity between both techniques is illustrated at multiple stages, including sample preparation, data collection and structural interpretation with a mechanistic perspective. The current state of instrumentation is described, focusing on synchrotron based setups. Present and future applications of Raman microspectrophotometry are reviewed with reference to recent examples dealing with metallo-, photosensitive-, and redox-proteins. The added value of Raman microspectrophotometry to assess X-radiation damage is discussed, and its applicability to investigate crystalline DNA molecules is also emphasized. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
    Full-text · Article · Jun 2011 · Biochimica et Biophysica Acta
  • Vincent Nivière · Florence Bonnot · Dominique Bourgeois

    No preview · Chapter · Dec 2010

Publication Stats

4k Citations
458.83 Total Impact Points

Institutions

  • 2004-2016
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2008-2014
    • University of Grenoble
      Grenoble, Rhône-Alpes, France
  • 2003-2012
    • University Joseph Fourier - Grenoble 1
      • Institut de Biologie Structurale
      Grenoble, Rhône-Alpes, France
  • 2010
    • University of Chicago
      Chicago, Illinois, United States
  • 2009-2010
    • Cea Leti
      Grenoble, Rhône-Alpes, France
    • The University of Manchester
      Manchester, England, United Kingdom
  • 1997-2009
    • European Synchrotron Radiation Facility
      Grenoble, Rhône-Alpes, France
  • 2005
    • Institut Pasteur
      Lutetia Parisorum, Île-de-France, France
  • 1998-1999
    • Institut de Biologie Structurale (IBS)
      Lutetia Parisorum, Île-de-France, France
    • Weizmann Institute of Science
      • Department of Structural Biology
      Rhovot, Central District, Israel