Dominique Bourgeois

University of Grenoble, Grenoble, Rhône-Alpes, France

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Publications (91)520.85 Total impact

  • [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.
    Current Opinion in Chemical Biology 06/2014; 20C:92-102. · 9.47 Impact Factor
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    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.
    PLoS ONE 01/2014; 9(6):e98362. · 3.53 Impact Factor
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    Chenxi Duan, Virgile Adam, Martin Byrdin, Dominique Bourgeois
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    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.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1148:177-202. · 1.29 Impact Factor
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    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.
    Current Opinion in Chemical Biology. 01/2014; 20:92–102.
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    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.
    Journal of the American Chemical Society 09/2013; · 10.68 Impact Factor
  • Dominique Bourgeois, Aline Regis-Faro, Virgile Adam
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    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.
    Biochemical Society Transactions 06/2012; 40(3):531-8. · 2.59 Impact Factor
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    Dominique Bourgeois, Virgile Adam
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    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.
    International Union of Biochemistry and Molecular Biology Life 04/2012; 64(6):482-91. · 2.79 Impact Factor
  • Arijit Roy, Martin J Field, Virgile Adam, Dominique Bourgeois
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    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.
    Journal of the American Chemical Society 11/2011; 133(46):18586-9. · 10.68 Impact Factor
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    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.
    Journal of the American Chemical Society 09/2011; 133(41):16362-5. · 10.68 Impact Factor
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    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.
    Biochimica et Biophysica Acta 06/2011; 1814(6):750-9. · 4.66 Impact Factor
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    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.
    Journal of Biophotonics 02/2011; 4(6):377-90. · 3.86 Impact Factor
  • Vincent Nivière, Florence Bonnot, Dominique Bourgeois
    Handbook of Metalloproteins, 12/2010; , ISBN: 9780470028636
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    ABSTRACT: X-ray-induced chemistry modifies biological macromolecules structurally and functionally, even at cryotemperatures. The mechanisms of x-radiation damage in colored or redox proteins have often been investigated by combining X-ray crystallography with in crystallo-ultraviolet-visible spectroscopy. Here, we used Raman microspectrophotometry to follow the onset of damage in crystalline lysozyme, notably that of disulfide bond breakage. The dose-dependent Raman spectra are consistent with a kinetic model for the rupture of disulfide bonds suggesting the rapid build up of an anionic radical intermediate. This intermediate may either revert back to the oxidized state or evolve toward protonated radical species or cleaved products. The data strongly suggest that back conversion of the anionic radical is significantly accelerated by X-rays, revealing an X-ray-induced "repair" mechanism. The possibility of X-ray-induced chemical repair is an important feature to take into account when assessing radiation damage in macromolecules.
    Structure 11/2010; 18(11):1410-9. · 5.99 Impact Factor
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    ABSTRACT: The fluorescent protein KillerRed generates reactive oxygen species through the CALI effect. This property paves the way for the design of genetically encoded photosensitizers for use in cell killing and cancer photodynamic therapy. In this article, we have investigated the diffusion pathways of di-oxygen and the superoxide radical in KillerRed, using molecular dynamics simulations. Our results suggest that, by comparison to the Ser-65-Thr mutant of GFP, diffusion of molecular oxygen (and singlet oxygen) is greatly facilitated in KillerRed, mostly due to the presence of a unique water-filled channel. In contrast, due to their negative charge, superoxide radical ions putatively produced inside the chromophore pocket are unable to escape the protein. These results are consistent with the hypothesis that superoxide generation, if it occurs, proceeds via light-induced photoreduction of the chromophore followed by long-range electron transfer, a mechanism in which the long hydrogen bond network through the channel could play a key role. Alternatively, the facilitated diffusion of di-oxygen through the channel suggests that singlet di-oxygen could be the principal cause of specific CALI of fused proteins. The entry of di-oxygen through the channel probably also accounts for the high susceptibility of KillerRed to photobleaching.
    Photochemical and Photobiological Sciences 10/2010; 9(10):1342-50. · 2.92 Impact Factor
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    ABSTRACT: 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 data storage both in 2D and 3D, using PCFPs and RSFPs. The irreversible burning of information was achieved by photoconverting from green to red defined areas in a layer of the PCFP Kaede. Data were also written and erased several times in layers of the photochromic fluorescent protein Dronpa. Using IrisFP, which combines the properties of PCFPs and RSFPs, we performed the first encoding of data in four colours using only one type of fluorescent protein. Finally, three-dimensional optical data storage was demonstrated using three mutants of EosFP (d1EosFP, mEosFP and IrisFP) in their crystalline form. Two-photon excitation allowed the precise addressing of regions of interest (ROIs) within the three-dimensional crystalline matrix without excitation of out-of-focus optical planes. Hence, this contribution highlights several data storage schemes based on the remarkable properties of PCFPs/RSFPs.
    Journal of Biotechnology 04/2010; 149(4):289-98. · 3.18 Impact Factor
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    ABSTRACT: 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 case of EYFP. The data suggest that photoinduced protonation of the chromophore is responsible for off-switching at cryo-temperature, and thus is possible in the absence of significant conformational freedom. This finding is consistent with the hypothesis that chromophore protonation may precede large amplitude conformational changes such as cis-trans isomerisation during off-photoswitching at room temperature. However, our data suggest that low-barrier photoinduced protonation pathways may in fact compete with room-temperature off-switching reactions in photochromic fluorescent proteins. The occurrence of reversible photoswitching at low-temperature is of interest to envisage cryo-nanoscopy experiments using genetically encoded fluorophores.
    Photochemical and Photobiological Sciences 02/2010; 9(2):254-62. · 2.92 Impact Factor
  • Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor
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    ABSTRACT: 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 illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of pi conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photobleaching in FPs, which are of importance in the rational design of photostable variants.
    Journal of the American Chemical Society 12/2009; 131(50):18063-5. · 10.68 Impact Factor
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    ABSTRACT: 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 this paper we present a theoretical investigation of the photoconversion reaction in the fluorescent protein EosFP, using excited-state hybrid quantum chemical and molecular mechanical potentials, in conjunction with reaction-path-finding techniques. Our results reveal a mechanism in which the hydroxybenzylidene moiety of the chromophore remains protonated and there is an excited state proton transfer from His62 to Phe61 that promotes peptide bond cleavage. Excitation of the neutral green form of EosFP to the first singlet excited state is followed by two intersystem crossing events, first to a triplet state and then back to the ground state singlet surface. From there, a number of rearrangements occur in the ground state and lead to the red form. Analyses of the structures and energies of the intermediates along the reaction path enable us to identify the critical role of the chromophore environment in promoting photoinduced backbone cleavage. Possible ways in which photoconvertible fluorescent proteins can be engineered to facilitate photoconversion are considered.
    Journal of the American Chemical Society 11/2009; 131(46):16814-23. · 10.68 Impact Factor
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    Dominique Bourgeois
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    ABSTRACT: In this issue of Structure, Knapp et al. show that allosteric changes in dimeric hemoglobin are retarded in the crystal, using time-resolved Laue diffraction. They observe that photo-dissociated carbon monoxide, after exploring a web of cavities, rebinds to the heme before it has a chance to exit the protein.
    Structure 11/2009; 17(11):1427-8. · 5.99 Impact Factor

Publication Stats

2k Citations
520.85 Total Impact Points

Institutions

  • 2014
    • University of Grenoble
      Grenoble, Rhône-Alpes, France
  • 2003–2012
    • University Joseph Fourier - Grenoble 1
      • • Laboratoire de Physiologie Cellulaire Végétale
      • • Institut de Biologie Structurale
      • • Laboratoire de Chimie et Biologie des Métaux
      Grenoble, Rhône-Alpes, France
  • 2011
    • University of Southampton
      Southampton, England, United Kingdom
  • 2009
    • University of Oxford
      • Department of Chemistry
      Oxford, ENG, United Kingdom
  • 2007–2009
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 1998–2009
    • Institut de Biologie Structurale (IBS)
      Lutetia Parisorum, Île-de-France, France
  • 1997–2009
    • European Synchrotron Radiation Facility
      Grenoble, Rhône-Alpes, France
  • 2005
    • University of Kansas
      Lawrence, Kansas, United States
  • 1997–2001
    • University of Chicago
      • Department of Biochemistry & Molecular Biology
      Chicago, IL, United States
  • 1996–1997
    • Architecture et Fonction des Macromolécules Biologiques
      Marsiglia, Provence-Alpes-Côte d'Azur, France