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20 Publications

Showing 11-20 of 20 results
08/08/18 | Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors.
Tebo AG, Pimenta FM, Zoumpoulaki M, Kikuti C, Sirkia H, Plamont M, Houdusse A, Gautier A
ACS Chemical Biology. 09/2018;13:2392 – 2397. doi: 10.1021/acschembio.8b00417

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

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

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

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

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

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

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

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05/02/17 | Discovery of chemoautotrophic symbiosis in the giant shipworm Kuphus polythalamia (Bivalvia: Teredinidae) extends wooden-steps theory
Distel DL, Altamia MA, Lin Z, Shipway JR, Han A, Forteza I, Antemano R, Limbaco MG, Tebo AG, Dechavez R, Albano J, Rosenberg G, Concepcion GP, Schmidt EW, Haygood MG
Proceedings of the National Academy of Sciences. 05/2017;114:E3652–E3658. doi: 10.1073/pnas.1620470114

Certain marine invertebrates harbor chemosynthetic bacterial symbionts, giving them the remarkable ability to consume inorganic chemicals such as hydrogen sulfide (H2S) rather than organic matter as food. These chemosynthetic animals are found near geochemical (e.g., hydrothermal vents) or biological (e.g., decaying wood or large animal carcasses) sources of H2S on the seafloor. Although many such symbioses have been discovered, little is known about how or where they originated. Here, we demonstrate a new chemosynthetic symbiosis in the giant teredinid bivalve (shipworm) Kuphus polythalamia and show that this symbiosis arose in a wood-eating ancestor via the displacement of ancestral cellulolytic symbionts by sulfur-oxidizing invaders. Here, wood served as an evolutionary stepping stone for a dramatic transition from heterotrophy to chemoautotrophy.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.

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02/08/17 | Intramolecular Photogeneration of a Tyrosine Radical in a Designed Protein
Tebo AG, Quaranta A, Herrero C, Pecoraro VL, Aukauloo A
ChemPhotoChem. 02/2017;1:89 – 92. doi: 10.1002/cptc.201600044

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.

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10/19/15 | Variable primary coordination environments of Cd(II) binding to three helix bundles provide a pathway for rapid metal exchange.
Tebo AG, Hemmingsen L, Pecoraro VL
Metallomics. 10/2015;7:1555 – 1561. doi: 10.1039/c5mt00228a

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.

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01/08/15 | Artificial metalloenzymes derived from three-helix bundles.
Tebo AG, Pecoraro VL
Current Opinion in Chemical Biology. 01/2015;25C:65 – 70. doi: 10.1016/j.cbpa.2014.12.034

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.

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08/01/08 | Porphyrins and Metalloporphyrins at Components in Artificial Photosynthesis Research
Tebo AG, Herrero C, Aukauloo A, Kadish KM, Smith KM, undefined , Guilard R
Handbook of Porphyrin Science;34:196 – 233. doi: 10.1142/9789814417297_0016
03/24/14 | Protein design: toward functional metalloenzymes.
Yu F, Cangelosi VM, Zastrow ML, Tegoni M, Plegaria JS, Tebo AG, Mocny CS, Ruckthong L, Qayyum H, Pecoraro VL
Chemical reviews. 03/2014;114:3495 – 3578. doi: 10.1021/cr400458x