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26 Publications

Showing 11-20 of 26 results
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    06/22/20 | A far‐red fluorescent chemogenetic reporter for in vivo molecular imaging
    Li C, Tebo AG, Thauvin M, Plamont M, Volovitch M, Morin X, Vriz S, Gautier A
    Angewandte Chemie International Edition. 06/2020:. doi: 10.1002/anie.202006576

    Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far‐red Fluorescence Activating and absorption Shifting Tag), a 14‐kDa monomeric protein that forms a bright far‐red fluorescent assembly with (4‐hydroxy‐3‐methoxy‐phenyl)allylidene rhodanine (HPAR‐3OM). As HPAR‐3OM is essentially non‐ fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR‐3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae and chicken embryo. Beyond enabling genetic encoding of far‐red fluorescence, frFAST allowed the design of a far‐ red chemogenetic reporter of protein‐protein interactions, demonstrating its great potential for the design of innovative far‐red emitting biosensors.

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    07/30/19 | Simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy
    Zhang R, Chouket R, Tebo AG, Plamont M, Kelemen Z, Gissot L, Faure J, Gautier A, Croquette V, Jullien L, Saux TL
    Optica. 07/2019;6:972. doi: 10.1364/optica.6.000972

    Fiber-optic epifluorescence imaging with one-photon excitation benefits from its ease of use, cheap light sources, and full-frame acquisition, which enables it for favorable temporal resolution of image acquisition. However, it suffers from a lack of robustness against autofluorescence and light scattering. Moreover, it cannot easily eliminate the out-of-focus background, which generally results in low-contrast images. In order to overcome these limitations, we have implemented fast out-of-phase imaging after optical modulation (Speed OPIOM) for dynamic contrast in fluorescence endomicroscopy. Using a simple and cheap optical-fiber bundle-based endomicroscope integrating modulatable light sources, we first showed that Speed OPIOM provides intrinsic optical sectioning, which restricts the observation of fluorescent labels at targeted positions within a sample. We also demonstrated that this imaging protocol efficiently eliminates the interference of autofluorescence arising from both the fiber bundle and the specimen in several biological samples. Finally, we could perform multiplexed observations of two spectrally similar fluorophores differing by their photoswitching dynamics. Such attractive features of Speed OPIOM in fluorescence endomicroscopy should find applications in bioprocessing, clinical diagnostics, plant observation, and surface imaging.

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    06/27/19 | A split fluorescent reporter with rapid and reversible complementation.
    Tebo AG, Gautier A
    Nature communications. 06/2019;10:2822. doi: 10.1038/s41467-019-10855-0

    Interactions between proteins play an essential role in metabolic and signaling pathways, cellular processes and organismal systems. We report the development of splitFAST, a fluorescence complementation system for the visualization of transient protein-protein interactions in living cells. Engineered from the fluorogenic reporter FAST (Fluorescence-Activating and absorption-Shifting Tag), which specifically and reversibly binds fluorogenic hydroxybenzylidene rhodanine (HBR) analogs, splitFAST displays rapid and reversible complementation, allowing the real-time visualization of both the formation and the dissociation of a protein assembly.

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    11/29/18 | Macroscale fluorescence imaging against autofluorescence under ambient light
    Zhang R, Chouket R, Plamont M, Kelemen Z, Espagne A, Tebo AG, Gautier A, Gissot L, Faure J, Jullien L, Croquette V, Saux TL
    Light: Science & Applications. 11/2018:1 – 12. doi: 10.1038/s41377-018-0098-6

    Macroscale fluorescence imaging is increasingly used to observe biological samples. However, it may suffer from spectral interferences that originate from ambient light or autofluorescence of the sample or its support. In this manuscript, we built a simple and inexpensive fluorescence macroscope, which has been used to evaluate the performance of Speed OPIOM (Out of Phase Imaging after Optical Modulation), which is a reference-free dynamic contrast protocol, to selectively image reversibly photoswitchable fluorophores as labels against detrimental autofluorescence and ambient light. By tuning the intensity and radial frequency of the modulated illumination to the Speed OPIOM resonance and adopting a phase-sensitive detection scheme that ensures noise rejection, we enhanced the sensitivity and the signal-to-noise ratio for fluorescence detection in blot assays by factors of 50 and 10, respectively, over direct fluorescence observation under constant illumination. Then, we overcame the strong autofluorescence of growth media that are currently used in microbiology and realized multiplexed fluorescence observation of colonies of spectrally similar fluorescent bacteria with a unique configuration of excitation and emission wavelengths. Finally, we easily discriminated fluorescent labels from the autofluorescent and reflective background in labeled leaves, even under the interference of incident light at intensities that are comparable to sunlight. The proposed approach is expected to find multiple applications, from biological assays to outdoor observations, in fluorescence macroimaging.

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    09/11/18 | Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy
    Tebo AG, Pimenta FM, Zhang Y, Gautier A
    Biochemistry. 11/2018;57:5648 – 5653. doi: 10.1021/acs.biochem.8b00649

    Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The fluorescence-activating and absorption shifting tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (so-called fluorogens). Here, we use rational design to modify the binding pocket of the protein and screen for improved fluorescence performances with four different fluorogens. The introduction of a single mutation results in improvements in both quantum yield and dissociation constant with nearly all fluorogens tested. Our improved FAST (iFAST) allowed the generation of a tandem iFAST (td-iFAST) that forms green and red fluorescent reporters 1.6-fold and 2-fold brighter than EGFP and mCherry, respectively, while having a comparable size.

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    08/28/18 | Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing
    Gautier A, Tebo AG
    BioEssays. 08/2018;40:1800118. doi: 10.1002/bies.201800118

    Fluorescence imaging has become an indispensable tool in cell and molecular biology. GFP‐like fluorescent proteins have revolutionized fluorescence microscopy, giving experimenters exquisite control over the localization and specificity of tagged constructs. However, these systems present certain drawbacks and as such, alternative systems based on a fluorogenic interaction between a chromophore and a protein have been developed. While these systems are initially designed as fluorescent labels, they also present new opportunities for the development of novel labeling and detection strategies. This review focuses on new labeling protocols, actuation methods, and biosensors based on fluorogenic protein systems. This review presents recently developed fluorogenic protein‐based systems made of a protein tag incorporating an external chromophore. Beyond addressing some limitations of classical fluorescent proteins, these unique systems present characteristics than can be used to creatively push the limits of biological imaging, in particular for the development of new labeling protocols, actuation methods and biosensors.

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    08/08/18 | Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors.
    Tebo AG, Pimenta FM, Zoumpoulaki M, Kikuti C, Sirkia H, Plamont M, Houdusse A, Gautier A
    ACS Chemical Biology. 09/2018;13:2392 – 2397. doi: 10.1021/acschembio.8b00417

    Fluorescent reporters are essential components for the design of optical biosensors that are able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca2+ in living mammalian cells in real time.

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    03/21/18 | Development of a Rubredoxin-Type Center Embedded in a de Dovo-Designed Three-Helix Bundle
    Tebo AG, Pinter TB, García-Serres R, Speelman AL, Tard C, Sénèque O, Blondin G, Latour J, Penner-Hahn J, Lehnert N, Pecoraro VL
    Biochemistry. 03/2018;57:2308 – 2316. doi: 10.1021/acs.biochem.8b00091

    Protein design is a powerful tool for interrogating the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements that are important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs normal hydrogen electrode). All previous attempts to design a rubredoxin site have focused on incorporating the canonical CXXC motifs in addition to reproducing the peptide fold or using flexible loop regions to define the morphology of the site. We have produced a rubredoxin site in an utterly different fold, a three-helix bundle. The spectra of this construct mimic the ultraviolet–visible, Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectra of native rubredoxin. Furthermore, the measured reduction potential suggests that this rubredoxin analogue could function similarly. Thus, we have shown that an α-helical scaffold sustains a rubredoxin site that can cycle with the desired potential between the Fe(II) and Fe(III) states and reproduces the spectroscopic characteristics of this electron transport protein without requiring the classic rubredoxin protein fold.

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    01/08/18 | Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity
    Koebke KJ, Yu F, Salerno E, Stappen CV, Tebo AG, Penner-Hahn JE, Pecoraro VL
    Angewandte Chemie International Edition. 01/2018;57:3954 – 3957. doi: 10.1002/anie.201712757

    Protein design is a useful strategy to interrogate the protein structure‐function relationship. We demonstrate using a highly modular 3‐stranded coiled coil (TRI‐peptide system) that a functional type 2 copper center exhibiting copper nitrite reductase (NiR) activity exhibits the highest homogeneous catalytic efficiency under aqueous conditions for the reduction of nitrite to NO and H2O. Modification of the amino acids in the second coordination sphere of the copper center increases the nitrite reductase activity up to 75‐fold compared to previously reported systems. We find also that steric bulk can be used to enforce a three‐coordinate CuI in a site, which tends toward two‐coordination with decreased steric bulk. This study demonstrates the importance of the second coordination sphere environment both for controlling metal‐center ligation and enhancing the catalytic efficiency of metalloenzymes and their analogues.

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    07/09/17 | Fluorogenic Labeling Strategies for Biological Imaging
    Li C, Tebo AG, Gautier A
    International Journal of Molecular Sciences. 07/2017;18:1473 – 11. doi: 10.3390/ijms18071473

    The spatiotemporal fluorescence imaging of biological processes requires effective tools to label intracellular biomolecules in living systems. This review presents a brief overview of recent labeling strategies that permits one to make protein and RNA strongly fluorescent using synthetic fluorogenic probes. Genetically encoded tags selectively binding the exogenously applied molecules ensure high labeling selectivity, while high imaging contrast is achieved using fluorogenic chromophores that are fluorescent only when bound to their cognate tag, and are otherwise dark. Beyond avoiding the need for removal of unbound synthetic dyes, these approaches allow the development of sophisticated imaging assays, and open exciting prospects for advanced imaging, particularly for multiplexed imaging and super-resolution microscopy.

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