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1. Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

Author

Hela Benaissa, Karim Ounoughi, Isabelle Aujard, Evelyne Fischer, Rosette Goïame, Julie Nguyen, Alison G. Tebo, Chenge Li, Thomas Le Saux, Giulia Bertolin, Marc Tramier, Lydia Danglot, Nicolas Pietrancosta, Xavier Morin, Ludovic Jullien, and Arnaud Gautier

Subjects
Science
Abstract

Fluorescent reporters spanning the visible spectrum are needed for imaging live cells and organisms. Here the authors report a collection of fluorogenic chromophores that cover the visible spectrum from blue to red using a single engineered and optimised protein tag.

Published
2021
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2. A split fluorescent reporter with rapid and reversible complementation

Author

Alison G. Tebo and Arnaud Gautier

Subjects
Science
Abstract

Monitoring protein-protein interactions via bimolecular fluorescence complementation is often limited by the slow kinetics and irreversibility of the complementation. Here the authors introduce a fluorescent reporter for real-time monitoring of reversible interactions based on complementation and binding of an exogenous chromophore.

Published
2019
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4. Fluorogenic Labeling Strategies for Biological Imaging

Author

Chenge Li, Alison G. Tebo, and Arnaud Gautier

Subjects
fluorogenic probes, protein labeling, RNA labeling, Biology (General), QH301-705.5, Chemistry, QD1-999
Abstract

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.

Published
2017
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5. The power of peer networking for improving STEM faculty job applications: a successful pilot programme

Author

Carlos M. Guardia, Erin Kane, Alison G. Tebo, Anna A. W. M. Sanders, Devrim Kaya, and Kathleen E. Grogan

Subjects
General Immunology and Microbiology, General Medicine, General Agricultural and Biological Sciences, General Biochemistry, Genetics and Molecular Biology, General Environmental Science
Abstract

To attain a faculty position, postdoctoral fellows submit job applications that require considerable time and effort to produce. Although mentors and colleagues review these applications, postdocs rarely receive iterative feedback from reviewers with the breadth of expertise typically found on an academic search committee. To address this gap, we describe an international peer-reviewing programme for postdocs across disciplines to receive reciprocal, iterative feedback on faculty applications. A participant survey revealed that nearly all participants would recommend the programme to others. Furthermore, our programme was more likely to attract postdocs who struggled to find mentoring, possibly because of their identity as a woman or member of an underrepresented population in STEM or because they changed fields. Between 2018 and 2021, our programme provided nearly 150 early career academics with a diverse and supportive community of peer mentors during the difficult search for a faculty position and continues to do so today. As the transition from postdoc to faculty represents the largest ‘leak’ in the academic pipeline, implementation of similar programmes by universities or professional societies would provide psycho-social support necessary to prevent attrition of individuals from underrepresented populations as well as increase the chances of success for early career academics in their search for independence.

Published
2023

6. Fluorescent chemigenetic actuators and indicators for use in living animals

Author

Helen Farrants and Alison G. Tebo

Subjects
Pharmacology, Drug Discovery, Animals, Humans, Protein Engineering, Fluorescent Dyes
Abstract

Fluorescent indicators and actuators provide a means to optically observe and perturb dynamic events in living animals. Although chemistry and protein engineering have contributed many useful tools to observe and perturb cells, an emerging strategy is to use chemigenetics: systems in which a small molecule dye interacts with a genetically encoded protein domain. Here we review chemigenetic strategies that have been successfully employed in living animals as photosensitizers for photoablation experiments, fluorescent cell cycle indicators, and fluorescent indicators for studying dynamic biological signals. Although these strategies at times suffer from challenges, e.g. delivery of the small molecule and assembly of the chemigenetic unit in living animals, the advantages of using small molecules with high brightness, low photobleaching, no chromophore maturation time and expanded color palette, combined with the ability to genetically target them to specific cell types, make chemigenetic fluorescent actuators and indicators an attractive strategy for use in living animals.

Published
2022

7. Nitrite reductase activity within an antiparallel de novo scaffold

Author

James E. Penner-Hahn, Vincent L. Pecoraro, Elizabeth C. Manickas, Karl J. Koebke, Alison G. Tebo, and Aniruddha Deb

Subjects
Coiled coil, Helix bundle, biology, Chemistry, Protein design, Active site, Antiparallel (biochemistry), Nitrite reductase, Biochemistry, Small molecule, Inorganic Chemistry, biology.protein, Biophysics, Binding site
Abstract

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis3 binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis3 binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis3 binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

Published
2021

8. Enhanced Photoinduced Electron Transfer Through a Tyrosine Relay in a De Novo Designed Protein Scaffold Bearing a Photoredox Unit and a Fe II S 4 Site

Author

Ally Aukauloo, Alison G. Tebo, Annamaria Quaranta, and Vincent L. Pecoraro

Subjects
chemistry.chemical_classification, Scaffold protein, Bearing (mechanical), Organic Chemistry, Protein design, Photoredox catalysis, Combinatorial chemistry, Photoinduced electron transfer, Analytical Chemistry, Amino acid, law.invention, Electron transfer, chemistry, law, Physical and Theoretical Chemistry, Tyrosine
Published
2021

9. The power of peer networking for improving STEM faculty job applications: a successful pilot program

Author

Kathleen E. Grogan, Carlos M. Guardia, Erin E. Kane, Anna A.W.M. Sanders, Devrim Kaya, and Alison G. Tebo

Subjects
Medical education, Job applications, education.field_of_study, ComputingMilieux_THECOMPUTINGPROFESSION, Population, medicine.disease, Power (social and political), medicine, Position (finance), Pilot program, Attrition, Early career, education, Psychology
Abstract

In order to successfully obtain a faculty position, postdoctoral fellows or ‘postdocs’, must submit an application which requires considerable time and effort to produce. These job applications are often reviewed by mentors and colleagues, but rarely are postdocs offered the opportunity to solicit feedback multiple times from reviewers with the same breadth of expertise often found on an academic search committee. To address this gap, this manuscript describes an international peer reviewing program for small groups of postdocs with a broad range of expertise to reciprocally and iteratively provide feedback to each other on their application materials. Over 145 postdocs have participated, often multiple times, over three years. A survey of participants in this program revealed that nearly all participants would recommend participation in such a program to other faculty applicants. Furthermore, this program was more likely to attract participants who struggled to find mentoring and support elsewhere, either because they changed fields or because of their identity as a woman or member of an underrepresented population in STEM. Participation in programs like this one could provide early career academics like postdocs with a diverse and supportive community of peer mentors during the difficult search for a faculty position. Such psychosocial support and encouragement has been shown to prevent attrition of individuals from these populations and programs like this one target the largest ‘leak’ in the pipeline, that of postdoc to faculty. Implementation of similar peer reviewing programs by universities or professional scientific societies could provide a valuable mechanism of support and increased chances of success for early-career academics in their search for independence.

Published
2021

10. Versatile On-Demand Fluorescent Labeling of Fusion Proteins Using Fluorescence-Activating and Absorption-Shifting Tag (FAST)

Author

Arnaud, Gautier, Ludovic, Jullien, Chenge, Li, Marie-Aude, Plamont, Alison G, Tebo, Marion, Thauvin, Michel, Volovitch, and Sophie, Vriz

Subjects
Microscopy, Fluorescence, Molecular Structure, Staining and Labeling, Recombinant Fusion Proteins, Animals, Humans, Flow Cytometry, Zebrafish, Cell Line, Fluorescent Dyes
Abstract

Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible.

Published
2021

11. Nitrite reductase activity within an antiparallel de novo scaffold

Author

Karl J, Koebke, Alison G, Tebo, Elizabeth C, Manickas, Aniruddha, Deb, James E, Penner-Hahn, and Vincent L, Pecoraro

Subjects
Electron Transport, Binding Sites, Nitrite Reductases, Catalytic Domain, Copper
Abstract

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis

Published
2021

12. Structure–function dataset reveals environment effects within a fluorescent protein model system

Author

Alison G. Tebo, Sam Duwé, Peter Dedecker, Luc Van Meervelt, Siewert Hugelier, Elke De Zitter, Wim Vandenberg, Groupe Dynamique et Cinétique des processus moléculaires (IBS-DYNAMOP), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Department of Chemistry [Leuven], Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Hasselt University (UHasselt), Janelia Research Campus [Ashburn] (HHMI Janelia), Howard Hughes Medical Institute (HHMI), European Project: 714688,NanoCellActivity, Tebo, Alison/0000-0003-0788-5617, Duwe, Sam/0000-0003-3768-1877, Van, Meervelt, Luc/0000-0003-2186-5209, Vandenberg, Wim/0000-0002-5888-9100, Dedecker, Peter/0000-0002-1882-2075, De Zitter, Elke, Hugelier, Siewert, DUWE, Sam, Vandenberg, Wim, Tebo, Alison G., Van Meervelt, Luc, and Dedecker, Peter

Subjects
Models, Molecular, Structure-Function Relationships, Polarity (physics), fluorescent proteins, Protein Conformation, Crystal structure, 010402 general chemistry, 01 natural sciences, Catalysis, biophysics, 0103 physical sciences, Molecule, function relationships, Spectroscopy, ComputingMilieux_MISCELLANEOUS, structure&#8211, 010304 chemical physics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010405 organic chemistry, Hydrogen bond, Chemistry, Hydrogen Bonding, General Chemistry, Protein engineering, General Medicine, photochromism, Fluorescence, Photochromism, 0104 chemical sciences, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Luminescent Proteins, Principal component analysis, Biological system
Abstract

Anisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure-function relationships. We examined this using fluorescent proteins as easy-to-modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods. We are grateful to Gerrit Groenhof (University of Jyvaskyla), Jeremy Harvey (KU Leuven), Raffaele Vitale (Universite de Lille), and Dominique Bourgeois (Institut de Biologie Structurale) for critical insights and discussion. E.D.Z. and L.V.M. thank the beamline staff from X06DA at the Swiss Light Source (Villigen, Switzerland), Proxima1 and Proxima2A at synchrotron Soleil (Gif-sur-Yvette, France), XRD1 at Elettra (Trieste, Italy) and I03 at Diamond Light Source (Oxfordshire, UK) for assistance during X-ray diffraction data collection. E.D.Z., S.H., and S.D. thank the Research Foundation Flanders (FWO) for a doctoral fellowship and postdoctoral fellowships (12X7919N and 12R2817N). This work was supported through funding from the Research Foundation Flanders through grants 1514319 N, G090819N, G0B8817N, and the European Research Council through grant 714688 NanoCellActivity. Dedecker, P (corresponding author), Katholieke Univ Leuven, Dept Chem, Celestijnenlaan 200G Box 2403, B-3001 Leuven, Belgium. peter.dedecker@kuleuven.be

Published
2021

13. Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

Author

Arnaud Gautier, Xavier Morin, Lydia Danglot, Alison G. Tebo, Julie Nguyen, Karim Ounoughi, Rosette Goïame, Thomas Le Saux, Chenge Li, Isabelle Aujard, Hela Benaissa, Nicolas Pietrancosta, Evelyne Fischer, Ludovic Jullien, Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des biomolécules (LBM UMR 7203), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de psychiatrie et neurosciences de Paris (IPNP - U1266 Inserm), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Janelia Research Campus [Ashburn] (HHMI Janelia), Howard Hughes Medical Institute (HHMI), Shangaï Jiao Tong University [Shangaï], Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Neurosciences Paris Seine (NPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), We thank Leducq establishment for funding the Leica SP8 Confocal/STED 3DX system, Sésame Région Ile-de-France for funding the Zeiss 880 Confocal/Airyscan system, and FLAG-ERA for grant SENSEÏ by ANR-19-HBPR-0003. This work has been supported by the European Research Council (ERC-2016-CoG-724705 FLUOSWITCH), the Agence Nationale de la Recherche (France BioImaging—ANR-10-INBS-04, Morphoscope2 - ANR-11-EQPX-0029, ANR-19-CE13-0026 ADOBE) and a prematuration grant from PSL University and QLife., ANR-19-HBPR-0003,SENSEI,Segmentation of Neurons using Standard and Super-Resolution Microscopy(2019), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-11-EQPX-0029,MORPHOSCOPE 2,Imagerie et reconstruction multiéchelles de la morphogenèse. (Plateforme d'innovation technologique et méthodologique pour l'imagerie in vivo et la reconstruction des dynamiques multiéchelles de la morphogenèse)(2011), ANR-19-CE13-0026,ADOBE,Dynamique de l'adhésion au niveau de l'enveloppe bactérienne(2019), European Project: ERC-2016-CoG-724705 ,FLUOSWITCH, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Chimie Moléculaire de Paris Centre (FR 2769), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Neuroscience Paris Seine (NPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Systèmes glutamatergiques normaux et pathologiques = Normal and Pathologic Glutamatergic Neurons (NPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Analyse, Interactions Moléculaires et Cellulaires (LBM-E2), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Chimie Moléculaire de Paris Centre (FR 2769), Gestionnaire, Hal Sorbonne Université, Segmentation of Neurons using Standard and Super-Resolution Microscopy - - SENSEI2019 - ANR-19-HBPR-0003 - FLAG-ERA JTC 2019 - VALID, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Equipements d'excellence - Imagerie et reconstruction multiéchelles de la morphogenèse. (Plateforme d'innovation technologique et méthodologique pour l'imagerie in vivo et la reconstruction des dynamiques multiéchelles de la morphogenèse) - - MORPHOSCOPE 22011 - ANR-11-EQPX-0029 - EQPX - VALID, Dynamique de l'adhésion au niveau de l'enveloppe bactérienne - - ADOBE2019 - ANR-19-CE13-0026 - AAPG2019 - VALID, and Pushing the frontiers of biological imaging with genetically encoded fluorescence switches - FLUOSWITCH - ERC-2016-CoG-724705 - INCOMING

Subjects
Diagnostic Imaging, Male, Materials science, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, Science, Green Fluorescent Proteins, Color, Biocompatible Materials, Nanotechnology, Biosensing Techniques, Protein tag, Protein Engineering, 010402 general chemistry, 01 natural sciences, Article, Fluorescence, Rats, Sprague-Dawley, 03 medical and health sciences, Fluorescence resonance energy transfer, Live cell imaging, Animals, Super-resolution microscopy, Coloring Agents, Fluorescent Dyes, 030304 developmental biology, Neurons, 0303 health sciences, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Rational design, STED microscopy, [CHIM.ORGA] Chemical Sciences/Organic chemistry, Directed evolution, Fusion protein, Rats, 0104 chemical sciences, 3. Good health, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, [CHIM.THEO] Chemical Sciences/Theoretical and/or physical chemistry, [SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging, Female, Electronics, Chemical tools, Visible spectrum
Abstract

Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we report the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. The ability to tune the fluorescence color and properties through simple molecular modulation provides a broad experimental versatility for imaging proteins in live cells, including neurons, and in multicellular organisms, and opens avenues for optimizing Förster resonance energy transfer (FRET) biosensors in live cells. The ability to tune the spectral properties and fluorescence performance enables furthermore to match the specifications and requirements of advanced super-resolution imaging techniques., Fluorescent reporters spanning the visible spectrum are needed for imaging live cells and organisms. Here the authors report a collection of fluorogenic chromophores that cover the visible spectrum from blue to red using a single engineered and optimised protein tag.

Published
2021

14. Versatile On-Demand Fluorescent Labeling of Fusion Proteins Using Fluorescence-Activating and Absorption-Shifting Tag (FAST)

Author

Sophie Vriz, Marie-Aude Plamont, Michel Volovitch, Chenge Li, Arnaud Gautier, Alison G. Tebo, Marion Thauvin, and Ludovic Jullien

Subjects
Fluorescent labelling, General method, Chemistry, On demand, Biophysics, Protein tag, Light Up, Absorption (electromagnetic radiation), Fusion protein, Fluorescence
Abstract

Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible.

Published
2021

15. Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins

Author

Elke De Zitter, Wim Vandenberg, Peter Dedecker, Luc Van Meervelt, Siewert Hugelier, Alison G. Tebo, and Sam Duwé

Subjects
Photochromism, Chemistry, Polarity (physics), Hydrogen bond, Molecule, Protein engineering, Chromophore, Biological system, Spectroscopy, Fluorescence
Abstract

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

Published
2020

16. A far-red fluorescent chemogenetic reporter for in vivo molecular imaging

Author

Chenge Li, Marion Thauvin, Arnaud Gautier, Xavier Morin, Sophie Vriz, Marie-Aude Plamont, Michel Volovitch, Alison G. Tebo, Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Shanghai Jiao Tong University [Shanghai], Laboratoire des biomolécules (LBM UMR 7203), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Paris (UP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), HAL-SU, Gestionnaire, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Chimie Moléculaire de Paris Centre (FR 2769), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), and Université Paris Cité (UPCité)

Subjects
far-red fluorescence, 0303 health sciences, High contrast, 010405 organic chemistry, Far-red, Deep tissue imaging, biosensors, 01 natural sciences, Fluorescence, 0104 chemical sciences, in vivo imaging, 03 medical and health sciences, chemistry.chemical_compound, Rhodanine, chemistry, In vivo, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Biophysics, chromophores, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular imaging, chemogenetic reporters, Biosensor, 030304 developmental biology
Abstract

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.

Published
2020

17. Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Author

Sophie Vriz, Irene Carlon-Andres, Dorothea Böken, Alison G. Tebo, Peter Dedecker, Benjamien Moeyaert, Arnaud Gautier, Marion Thauvin, Michel Volovitch, Sergi Padilla-Parra, HAL-SU, Gestionnaire, Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Technologie campus Gent - KU Leuven (KU Leuven), Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Oxford, Université Paris sciences et lettres (PSL), King‘s College London, Université Paris Cité (UPCité), Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), University of Oxford [Oxford], and Université de Paris (UP)

Subjects
Fluorophore, Cell division, Oligonucleotides, Color, Gene Expression, Biosensing Techniques, Saccharomyces cerevisiae, MOLECULAR BIOLOGY METHODS, Protein Engineering, Benzylidene Compounds, 01 natural sciences, Article, Imaging, 03 medical and health sciences, chemistry.chemical_compound, Orthogonality, Live cell imaging, Chlorocebus aethiops, Escherichia coli, Fluorescence microscope, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cloning, Molecular, Molecular Biology, Zebrafish, Fluorescent Dyes, 030304 developmental biology, Physics, 0303 health sciences, Staining and Labeling, 010405 organic chemistry, Chemistry, 030302 biochemistry & molecular biology, Optical Imaging, A protein, Cell Biology, Protein engineering, Chromophore, Fluorescence, Cell function, Recombinant Proteins, 0104 chemical sciences, COS Cells, Biological system, Chemical tools, Plasmids
Abstract

Fluorescence microscopy is an indispensable tool in biological research, allowing sub-second and sub-micrometer mapping of molecules or processes inside living cells. Moreover, using spectrally separated fluorophores, one can observe multiple targets simultaneously, leading to a deeper understanding of the dynamic molecular interplays that regulate cell function and fate. Chemogenetic systems, which combine a protein tag and a synthetic fluorophore, provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. However, the fluorophore promiscuity of chemogenetic systems renders two-color applications challenging. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. A two-color cell cycle sensor based on greenFAST and redFAST is capable of detecting very short, early cell cycles in zebrafish development which had previously been difficult to image. Furthermore, this pair of orthogonal tags can be developed into split complementation systems that are capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy.

Published
2020

18. Sensing cellular biochemistry with fluorescent chemical-genetic hybrids

Author

Alison G. Tebo, Arnaud Gautier, Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), HAL-SU, Gestionnaire, Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), and Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Fluorescence-lifetime imaging microscopy, Nanotechnology, macromolecular substances, Biosensing Techniques, Fluorescent chemical–genetic hybrids, 010402 general chemistry, 01 natural sciences, Biochemistry, Fluorescence imaging, Analytical Chemistry, 03 medical and health sciences, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Interaction Maps, Fluorescent Dyes, Chemistry, Biosensing, Optical Imaging, Proteins, Fluorescence, 0104 chemical sciences, 030104 developmental biology, Semisynthetic fluorescent sensors, RNA, Spatiotemporal resolution, Biosensor, Macromolecule
Abstract

International audience; Fluorescent biosensors are powerful tools with which to detect biochemical events inside of cells with high spatiotemporal resolution. Biosensors based on fluorescent proteins often suffer from issues with photostability and brightness. On the other hand, hybrid, chemical-genetic systems present unique opportunities to combine the strengths of synthetic, organic chemistry with biological macromolecules to generate exquisitely tailored semisynthetic sensors.

Published
2019

19. Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity

Author

Elvin V. Salerno, Fangting Yu, Casey Van Stappen, Karl J. Koebke, Alison G. Tebo, Vincent L. Pecoraro, and James E. Penner-Hahn

Subjects
Steric effects, Nitrite Reductases, Coordination sphere, Protein design, chemistry.chemical_element, Protein Engineering, 010402 general chemistry, 01 natural sciences, Article, Catalysis, chemistry.chemical_compound, Amino Acid Sequence, Nitrite, Coiled coil, Binding Sites, 010405 organic chemistry, General Medicine, General Chemistry, Protein engineering, Nitrite reductase, Combinatorial chemistry, Copper, Protein Structure, Tertiary, 0104 chemical sciences, Kinetics, X-Ray Absorption Spectroscopy, chemistry, Biocatalysis, Mutagenesis, Site-Directed, Peptides
Abstract

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 H2 O. 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.

Published
2018

20. Light-driven electron transfer in a modular assembly of a ruthenium(II) polypyridine sensitiser and a manganese(II) terpyridine unit separated by a redox active linkage. DFT analysis

Author

Stefano Protti, Ally Aukauloo, Rajaa Farran, Shyamal Das, Winfried Leibl, Reza Fallahpour, Annamaria Quaranta, Christian Herrero, Alison G. Tebo, and Marie-France Charlot

Subjects
010405 organic chemistry, General Chemical Engineering, chemistry.chemical_element, General Chemistry, Chromophore, 010402 general chemistry, Photochemistry, 01 natural sciences, Redox, 0104 chemical sciences, law.invention, Ruthenium, Artificial photosynthesis, Bipyridine, chemistry.chemical_compound, Electron transfer, chemistry, law, Terpyridine, Electron paramagnetic resonance
Abstract

A series of ruthenium polypyridine-based complexes covalently bound to a terpyridine coordinating site for MnII ion coordination has been developed. A redox active unit separates the photoactive unit and the manganese complex. Introducing ester groups on the bipyridine skeleton allows modulation of redox properties of the chromophore. Intramolecular electron transfer from the MnII to the photogenerated RuIII was studied by time-resolved transient absorption and EPR. Photophysical studies support the participation of the imidazole unit in the electron transfer process from the Mn(II) complex and Ru(III) in the case of ester containing chromophores. DFT calculations were performed and used to rationalize the photophysical behavior of the complexes, in particular the effect of coordination of the MnII ion to the terpyridine cavity as well as the influence of the electron withdrawing groups on the Ru chromophore.

Published
2017

21. Out-of-Phase Imaging after Optical Modulation for Micro-and Macro-scale Multiplexed Fluorescence Imaging Against Autofluorescence and Ambient Light

Author

Jérôme Quérard, Raja Chouket, Lionel Gissot, Ruikang Zhang, Alison G. Tebo, Vincent Croquette, Marie-Aude Plamont, Thomas Le Saux, Arnaud Gautier, Ludovic Jullien, Zsolt Kelemen, Jean-Denis Faure, and Agathe Espagne

Subjects
Autofluorescence, Out of phase, Fluorescence-lifetime imaging microscopy, Materials science, Macroscopic scale, business.industry, Modulation, Optoelectronics, business, Multiplexing
Published
2019

22. Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors

Author

Alison G. Tebo, Carlos Kikuti, Martha Zoumpoulaki, Marie-Aude Plamont, Helena Sirkia, Arnaud Gautier, Frederico M. Pimenta, Anne Houdusse, Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Compartimentation et dynamique cellulaires (CDC), and Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Analyte, Rhodanine, Biosensing Techniques, macromolecular substances, Ligands, Photoreceptors, Microbial, 010402 general chemistry, 01 natural sciences, Biochemistry, Fluorescence, Bacterial Proteins, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Fluorescent Dyes, 010405 organic chemistry, business.industry, Chemistry, Optical Imaging, technology, industry, and agriculture, General Medicine, Modular design, 0104 chemical sciences, Microscopy, Fluorescence, Biophysics, Molecular Medicine, Calcium, business, Biosensor, HeLa Cells, Protein Binding
Abstract

International audience; 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.

Published
2018
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23. Development of a rubredoxin-type center embedded in a de novo designed three-helix bundle

Author

Tyler B. J. Pinter, Jean-Marc Latour, Vincent L. Pecoraro, Geneviève Blondin, Alison G. Tebo, Nicolai Lehnert, Ricardo Garcia-Serres, Cédric Tard, Olivier Sénèque, Amy L. Speelman, James E. Penner-Hahn, University of Michigan [Ann Arbor], University of Michigan System, Department of Chemistry and Biophysics, University of Michigan, University of Michigan System-University of Michigan System, Laboratoire de Chimie et Biologie des Métaux (LCBM - UMR 5249), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de chimie moléculaire (LCM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X), ANR-11-LABX-0003,ARCANE,Grenoble, une chimie bio-motivée(2011), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and École polytechnique (X)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Protein Conformation, alpha-Helical, Circular dichroism, Iron, Protein design, 010402 general chemistry, Ferric Compounds, 01 natural sciences, Biochemistry, Article, law.invention, Electron Transport, Electron transfer, Protein structure, law, Rubredoxin, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Electron paramagnetic resonance, Helix bundle, 010405 organic chemistry, Magnetic circular dichroism, Chemistry, Circular Dichroism, Rubredoxins, Electron Spin Resonance Spectroscopy, 0104 chemical sciences, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Crystallography, Oxidation-Reduction
Abstract

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.

Published
2018

24. Artificial metalloenzymes derived from three-helix bundles

Author

Vincent L. Pecoraro and Alison G. Tebo

Subjects
chemistry.chemical_classification, Primary (chemistry), Coordination sphere, Chemistry, Stereochemistry, Biochemistry, Article, Protein Structure, Secondary, Enzymes, Analytical Chemistry, Zinc, Drug Design, Metalloproteins, Helix, Metalloprotein, Copper
Abstract

Three-helix bundles and coiled-coil motifs are well-established de novo designed scaffolds that have been investigated for their metal-binding and catalytic properties. Satisfaction of the primary coordination sphere for a given metal is sufficient to introduce catalytic activity and a given structure may catalyze different reactions dependent on the identity of the incorporated metal. Here we describe recent contributions in the de novo design of metalloenzymes based on three-helix bundles and coiled-coil motifs, focusing on non-heme systems for hydrolytic and redox chemistry.

Published
2015

25. Intramolecular Photogeneration of a Tyrosine Radical in a Designed Protein

Author

Christian Herrero, Alison G. Tebo, Vincent L. Pecoraro, Ally Aukauloo, Annamaria Quaranta, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Photocatalyse et Biohydrogène (LPB), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
photochemistry, 010405 organic chemistry, Chemistry, Charge separation, Radical, Tyrosine radical, [SDV]Life Sciences [q-bio], Organic Chemistry, Protein design, 010402 general chemistry, Photochemistry, radicals, 01 natural sciences, Article, 0104 chemical sciences, Analytical Chemistry, Electron transfer, Intramolecular force, Tyrosine, Physical and Theoretical Chemistry, protein design
Abstract

Long-distance biological electron transfer occurs through a hopping mechanism and often involves tyrosine as a high potential intermediate, for example in the early charge separation steps during photosynthesis. Protein design allows for the development of minimal systems to study the underlying principles of complex systems. Herein, we report the development of the first ruthenium-linked designed protein for the photogeneration of a tyrosine radical by intramolecular electron transfer.

Published
2017

26. Discovery of chemoautotrophic symbiosis in the giant shipworm

Author

Daniel L, Distel, Marvin A, Altamia, Zhenjian, Lin, J Reuben, Shipway, Andrew, Han, Imelda, Forteza, Rowena, Antemano, Ma Gwen J Peñaflor, Limbaco, Alison G, Tebo, Rande, Dechavez, Julie, Albano, Gary, Rosenberg, Gisela P, Concepcion, Eric W, Schmidt, and Margo G, Haygood

Subjects
Chemoautotrophic Growth, Bacteria, Animals, Symbiosis, Wood, Bivalvia
Abstract

The "wooden-steps" hypothesis [Distel DL, et al. (2000)

Published
2017

27. Discovery of chemoautotrophic symbiosis in the giant shipworm Kuphus polythalamia (Bivalvia: Teredinidae) extends wooden-steps theory

Author

Gary Rosenberg, Margo G. Haygood, Julie Albano, Andrew W. Han, Zhenjian Lin, J. Reuben Shipway, Daniel L. Distel, Rowena R. Antemano, Rande Dechavez, Ma. Gwen J. Peñaflor Limbaco, Gisela P. Concepcion, Eric W. Schmidt, Imelda Forteza, Alison G. Tebo, and Marvin A. Altamia

Subjects
0106 biological sciences, 0301 basic medicine, Shipworms, 010603 evolutionary biology, 01 natural sciences, 03 medical and health sciences, Symbiosis, 14. Life underwater, Biology, Chemosynthesis, Multidisciplinary, biology, Phylogenetic tree, Endosymbiosis, Ecology, Bivalvia, biology.organism_classification, symbiosis, 030104 developmental biology, Habitat, shipworm, thioautotrophy, Teredinidae, chemoautotrophy, Hydrothermal vent
Abstract

The “wooden-steps” hypothesis [Distel DL, et al. (2000) Nature 403:725–726] proposed that large chemosynthetic mussels found at deep-sea hydrothermal vents descend from much smaller species associated with sunken wood and other organic deposits, and that the endosymbionts of these progenitors made use of hydrogen sulfide from biogenic sources (e.g., decaying wood) rather than from vent fluids. Here, we show that wood has served not only as a stepping stone between habitats but also as a bridge between heterotrophic and chemoautotrophic symbiosis for the giant mud-boring bivalve Kuphus polythalamia. This rare and enigmatic species, which achieves the greatest length of any extant bivalve, is the only described member of the wood-boring bivalve family Teredinidae (shipworms) that burrows in marine sediments rather than wood. We show that K. polythalamia harbors sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria instead of the cellulolytic symbionts that allow other shipworm species to consume wood as food. The characteristics of its symbionts, its phylogenetic position within Teredinidae, the reduction of its digestive system by comparison with other family members, and the loss of morphological features associated with wood digestion indicate that K. polythalamia is a chemoautotrophic bivalve descended from wood-feeding (xylotrophic) ancestors. This is an example in which a chemoautotrophic endosymbiosis arose by displacement of an ancestral heterotrophic symbiosis and a report of pure culture of a thioautotrophic endosymbiont.

Published
2017

28. Protein Design: Toward Functional Metalloenzymes

Author

Vincent L. Pecoraro, Hira Qayyum, Virginia M. Cangelosi, Alison G. Tebo, Jefferson S. Plegaria, Melissa L. Zastrow, Catherine S. Mocny, Matteo Tegoni, Leela Ruckthong, and Fangting Yu

Subjects
Adenosine Deaminase, Chemistry, Protein design, Zinc Fingers, DNA, Heme, General Chemistry, Alkenes, Protein Engineering, Article, Protein Structure, Tertiary, Biochemistry, Metals, Heavy, Metalloproteins, Animals, Protein Binding
Published
2014

29. Simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy

Author

Jean-Denis Faure, Alison G. Tebo, Thomas Le Saux, Ruikang Zhang, Zsolt Kelemen, Raja Chouket, Vincent Croquette, Arnaud Gautier, Marie-Aude Plamont, Ludovic Jullien, Lionel Gissot, Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Jean-Pierre Bourgin (IJPB), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Université Paris-Saclay, Laboratoire de Physique Statistique de l'ENS (LPS), Fédération de recherche du Département de physique de l'Ecole Normale Supérieure - ENS Paris (FRDPENS), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Agence Nationale de la Recherche (ANR)French National Research Agency (ANR) [ANR10-LABX-0040-SPS, France BioImaging-ANR-10-INBS-04, Morphoscope2-ANR-11-EQPX-0029, ANR-11-IDEX-0003-02], Fondation pour la Recherche Medicale (FRM)Fondation pour la Recherche Medicale, Mission Interdisciplinarite du CNRS, Domaine d'Interet Majeur Analytics de la Region Ile de France (DREAM), SATT Lutech (OPIOM), Gestionnaire, Hal Sorbonne Université, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
spectroscopy, Optical sectioning, Computer science, [SDV]Life Sciences [q-bio], fibers, 01 natural sciences, Multiplexing, Light scattering, 010309 optics, 03 medical and health sciences, Optics, 0103 physical sciences, Endomicroscopy, Fluorescence microscope, 030304 developmental biology, 0303 health sciences, business.industry, endoscope, tissue, Fluorescence, proteins, Atomic and Molecular Physics, and Optics, Electronic, Optical and Magnetic Materials, [SDV] Life Sciences [q-bio], Autofluorescence, confocal, Temporal resolution, business
Abstract

International audience; 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.

Published
2019

30. Variable primary coordination environments of Cd(II) binding to three helix bundles provide a pathway for rapid metal exchange

Author

Lars Hemmingsen, Vincent L. Pecoraro, and Alison G. Tebo

Subjects
Coordination sphere, Stereochemistry, Protein design, Molecular Sequence Data, Biophysics, Plasma protein binding, Biochemistry, Article, Biomaterials, Metal, Bacterial Proteins, Metalloproteins, Metalloprotein, Amino Acid Sequence, Peptide sequence, chemistry.chemical_classification, Metals and Alloys, Nuclear magnetic resonance spectroscopy, chemistry, Chemistry (miscellaneous), visual_art, Helix, visual_art.visual_art_medium, Sequence Alignment, Cadmium, Protein Binding
Abstract

Members of the ArsR/SmtB family of transcriptional repressors, such as CadC, regulate the intracellular levels of heavy metals like Cd(II), Hg(II), and Pb(II). These metal sensing proteins bind their target metals with high specificity and affinity, however, a lack of structural information about these proteins makes defining the coordination sphere of the target metal difficult. Lingering questions as to the identity of Cd(II) coordination in CadC are addressed via protein design techniques. Two designed peptides with tetrathiolate metal binding sites were prepared and characterized, revealing fast exchange between CdS3O and CdS4 coordination spheres. Correlation of (111m)Cd PAC spectroscopy and (113)Cd NMR spectroscopy suggests that Cd(II) coordinated to CadC is in fast exchange between CdS3O and CdS4 forms, which may provide a mechanism for rapid sensing of heavy metal contaminants by this regulatory protein.

Published
2015

32. Improved Chemical-Genetic Fluorescent Markers for Live Cell Microscopy

Author

Alison G. Tebo, Yu Zhang, Frederico M. Pimenta, Arnaud Gautier, Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Light, Rhodanine, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, Cell, Photoreceptors, Microbial, Biochemistry, Fluorescence, Green fluorescent protein, 03 medical and health sciences, Bacterial Proteins, Live cell imaging, Microscopy, Escherichia coli, medicine, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Fluorescent Dyes, Binding Sites, Microscopy, Confocal, Photobleaching, Chemistry, Rational design, Halorhodospira halophila, Dissociation constant, HEK293 Cells, Spectrometry, Fluorescence, 030104 developmental biology, medicine.anatomical_structure, Mutation, Mutagenesis, Site-Directed, Biophysics, mCherry, Protein Binding
Abstract

International audience; 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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