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30 Publications

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    01/01/24 | Transforming chemigenetic bimolecular fluorescence complementation systems into chemical dimerizers using chemistry.
    Pratik Kumar , Alina Gutu , Amelia Waring , Timothy A. Brown , Luke D. Lavis , Alison G. Tebo
    bioRxiv. 2024 Jan 01:. doi: 10.1101/2023.12.30.573644

    Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

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    11/29/23 | A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction
    Xiao Li , Rico Gamuyao , Ming-Lun Wu , Woo Jung Cho , Nathan B. Kurtz , Sharon V. King , R.A. Petersen , Daniel R. Stabley , Caleb Lindow , Leslie Climer , Abbas Shirinifard , Francesca Ferrara , Robert E. Throm , Camenzind G. Robinson , Alex Carisey , Alison G. Tebo , Chi-Lun Chang
    bioRxiv. 2023 Nov 29:. doi: 10.1101/2023.11.29.569289

    Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen- Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

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    10/18/23 | A blue-shifted genetically encoded Ca2+ indicator with enhanced two-photon absorption
    Abhi Aggarwal , Smrithi Sunil , Imane Bendifallah , Michael Moon , Mikhail Drobizhev , Landon Zarowny , Jihong Zheng , Sheng-Yi Wu , Alexander W. Lohman , Alison G. Tebo , Valentina Emiliani , Kaspar Podgorski , Yi Shen , Robert E. Campbell
    bioRxiv. 2023 Oct 18:. doi: 10.1101/2023.10.12.562058

    Significance Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.

    Aim We describe the development and applications of T-GECO1 – a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1.

    Approach We used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices.

    Results The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.

    Conclusion T-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

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    06/27/23 | Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging.
    Usama SM, Marker SC, Li D, Caldwell DR, Stroet M, Patel NL, Tebo AG, Hernot S, Kalen JD, Schnermann M
    Journal of the American Chemical Society. 2023 Jun 27;145(27):14647–14659. doi: 10.1021/jacs.3c01765

    Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

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    01/15/22 | Fluorescent chemigenetic actuators and indicators for use in living animals.
    Farrants H, Tebo AG
    Current Opinion in Pharmacology. 2022 Jan 15;62:159-167. doi: 10.1016/j.coph.2021.12.007

    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.

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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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