Alison Tebo

Janelia Farm Research Campus · Molecular Tools and Imaging

Protein-protein interactions (PPI) can be detected through selective complementation of split fluorescent reporters made of two complementary fragments that reassemble into a functional fluorescent reporter when in close proximity. We previously introduced splitFAST, a chemogenetic PPI reporter with rapid and reversible complementation. Here, we pr...

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5–9.2-fold, can be e...

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...

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 p...

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 chromophor...

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 sym...

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,...

Electron transfer 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 de novo protei...

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...

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...

Fluorescent reporters are essential tools in cell biology for imaging the dynamics of proteins in living cells and organisms with high spatial and temporal resolution. Chemogenetic systems made of a genetically encoded protein tag acting as an anchor for synthetic fluorophores combine the targeting selectivity of genetic tags with the advantages of...

Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no re...

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...

Fluorescent biosensors are powerful tools for the detection of biochemical events inside 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 synthet...

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‐...

Far‐red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we report a far‐red fluorescent chemogenetic reporter for the on‐demand imaging of fusion proteins in cells and in living multicellular organisms. Abstract Far‐red emitting fluorescent labels are highly desirable for spectral multiplex...

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...

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-...

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...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Interactions between proteins play an essential role in metabolic and signaling pathways, cellular processes and organismal systems. We report the development of splitFAST, a fluorescence complementation system for the visualization of transient protein-protein interactions in living cells. Engineered from the fluorogenic reporter FAST (Fluorescenc...

In micro-and macro-scale fluorescence imaging, Speed Out-of-Phase Imaging after Optical Modulation (Speed OPIOM) is shown to be highly efficient for multiplexed fluorescence imaging in the presence of autofluorescence and ambient light.

Fluorescence imaging at the macroscale will become easier and more sensitive thanks to a new optical procedure, which uses photoswitchable fluorescent tags. Until now, observing signals from the fluorescent labels has been hampered by interference due to external light or the natural fluorescence of other chemicals in a biological sample or in the...

Inducible chemical-genetic fluorescent markers are promising tools for live cell imaging requiring high spatiotemporal resolution and low background fluorescence. The Fluorescence-Activating and ab-sorption Shifting Tag (FAST) was recently developed to form fluorescent molecular complexes with a family of small, synthetic fluorogenic chromophores (...

Fluorescence imaging has become an indispensable tool in cell and molecular biology. GFP-like fluorescent proteins have revolutionized fluorescence microscopy, giving experimenters exquisite control over the localization and specificity of tagged constructs. However, these systems present certain drawbacks and as such, alternative systems based on...

Protein design is a powerful tool to interrogate the basic requirements for the function of a metal site in a way that allows for the selective incorporation of elements important for function. Rubredoxins are small electron transfer proteins with a reduction potential centered near 0 mV (vs. NHE). All previous attempts at the design of a rubredoxi...

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...

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 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 bin...

Significance Certain marine invertebrates harbor chemosynthetic bacterial symbionts, giving them the remarkable ability to consume inorganic chemicals such as hydrogen sulfide (H 2 S) 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 ani...

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...

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. Intramolecula...

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 spher...

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 t...

Protein design is a powerful way to interrogate the basic requirements for function of metal sites by systematically incorporating elements important for function. Single-stranded three-helix bundles with either thiolate-rich sites for spectroscopic characterization and electron transfer, or histidine-rich sites for redox catalysis are described....

Protein design is a valuable tool for understanding the fundamental factors that dictate protein structure and function. The field of protein design has seen significant progress with a growing range of applications in areas of research from fundamental biochemistry to biotechnology and medicine. Rapid advancement of computer applications in biotec...