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6 Publications

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    11/30/21 | Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging.
    Benaissa H, Ounoughi K, Aujard I, Fischer E, Goïame R, Nguyen J, Tebo AG, Li C, Le Saux T, Bertolin G, Tramier M, Danglot L, Pietrancosta N, Morin X, Jullien L, Gautier A
    Nature Communications. 2021 Nov 30;12(1):6989. doi: 10.1038/s41467-021-27334-0

    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.

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    10/31/21 | Versatile On-Demand Fluorescent Labeling of Fusion Proteins Using Fluorescence-Activating and Absorption-Shifting Tag (FAST).
    Gautier A, Jullien L, Li C, Plamont M, Tebo AG, Thauvin M, Volovitch M, Vriz S
    Methods Mol Biol. 2021;2350:253-265. doi: 10.1007/978-1-0716-1593-5_16

    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.

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    10/18/21 | The power of peer networking for improving STEM faculty job applications: a successful pilot program
    Guardia CM, Kane E, Tebo AG, Sanders AA, Kaya D, Grogan KE
    bioRxiv. 10/2021:. doi: 10.1101/2021.10.16.464662

    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.Competing Interest StatementThe authors have declared no competing interest.

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    09/06/21 | Nitrite reductase activity within an antiparallel de novo scaffold.
    Koebke KJ, Tebo AG, Manickas EC, Deb A, Penner-Hahn JE, Pecoraro VL
    JBIC Journal of Biological Inorganic Chemistry. 09/2021;26(7):855 - 862. doi: 10.1007/s00775-021-01889-1

    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.

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    03/15/21 | Enhanced Photoinduced Electron Transfer Through a Tyrosine Relay in a De Novo Designed Protein Scaffold Bearing a Photoredox Unit and a Fe <sup>II</sup> S <sub>4</sub> Site
    Tebo A, Quaranta A, Pecoraro VL, Aukauloo A
    ChemPhotoChem. 03/2021;5(7):665 - 668. doi: 10.1002/cptc.v5.710.1002/cptc.202100014

    Electron transfer (ET) processes in biology over long distances often proceed via a series of hops, which reduces the distance dependence of the rate of ET. The protein matrix itself can be involved in mediating ET directly through the participation of redox-active amino acids. We have designed an electron transfer chain incorporated into a de novo protein scaffold, which is capable of photoinduced intramolecular electron transfer between a photoredox unit and a FeIIS4 site through a tyrosine amino acid relay. The kinetics were characterized by nanosecond laser pulse photolysis and revealed that electron transfer from [RuIIIbpymal]3+ proceeds most efficiently via a tyrosine located ∼16 Å from Rubpymal (bpymal=1-((1-([2,2′-bipyridin]-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-1H-pyrrole-2,5-dione). Removal of the tyrosine as the electron relay station results in a 20-fold decrease in the apparent rate constant for the electron transfer.

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    02/04/21 | Structure-Function Dataset Reveals Environment Effects within a Fluorescent Protein Model System.
    De Zitter E, Hugelier S, Duwé S, Vandenberg W, Tebo AG, Van Meervelt L, Dedecker P
    Angew Chemie (International Edition English). 2021 Feb 04:. doi: 10.1002/anie.202015201

    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.

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