LanTERN: A Fluorescent Sensor That Specifically Responds to Lanthanides

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This article references 16 other publications.

1. 1

   Cheisson, T.; Schelter, E. J. Rare Earth Elements: Mendeleev’s Bane, Modern Marvels. Science 2019, 363 (6426), 489– 493,  DOI: 10.1126/science.aau7628

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   Rare earth elements: Mendeleev's bane, modern marvels

   Cheisson, Thibault; Schelter, Eric J.

   Science (Washington, DC, United States) (2019), 363 (6426), 489-493CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

   A review. The rare earths (REs) are a family of 17 elements that exhibit pronounced chem. similarities as a group, while individually expressing distinctive and varied electronic properties. These atomistic electronic properties are extraordinarily useful and motivate the application of REs in many technologies and devices. From their discovery to the present day, a major challenge faced by chemists has been the sepn. of RE elements, which has evolved from tedious crystn. to highly engineered solvent extn. schemes. The increasing incorporation and dependence of REs in technol. have raised concerns about their sustainability and motivated recent studies for improved sepns. to achieve a circular RE economy.
2. 2

   Pol, A.; Barends, T. R. M.; Dietl, A.; Khadem, A. F.; Eygensteyn, J.; Jetten, M. S. M.; Op den Camp, H. J. M. Rare Earth Metals Are Essential for Methanotrophic Life in Volcanic Mudpots. Environ. Microbiol. 2014, 16 (1), 255– 264,  DOI: 10.1111/1462-2920.12249

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   Rare earth metals are essential for methanotrophic life in volcanic mudpots

   Pol, Arjan; Barends, Thomas R. M.; Dietl, Andreas; Khadem, Ahmad F.; Eygensteyn, Jelle; Jetten, Mike S. M.; Op den Camp, Huub J. M.

   Environmental Microbiology (2014), 16 (1), 255-264CODEN: ENMIFM; ISSN:1462-2912. (Wiley-Blackwell)

   Growth of Methylacidiphilum fumariolicum SolV, an extremely acidophilic methanotrophic microbe isolated from an Italian volcanic mudpot, is strictly dependent on the presence of lanthanides, a group of rare earth elements (REEs) such as lanthanum (Ln), cerium (Ce), praseodymium Pr and neodymium (Nd). After fractionation of the bacterial cells and crystn. of the methanol dehydrogenase (MDH),it was shown that lanthanides were essential as cofactor in a homodimeric MDH comparable with one of the MDHs of Methylobacterium extorquens AM1. The authors hypothesize that the lanthanides provide superior catalytic properties to pyrroloquinoline quinone (PQQ)-dependent MDH, which is a key enzyme for both methanotrophs and methylotrophs. Thus far, all isolated MxaF-type MDHs contain calcium as a catalytic cofactor. The gene encoding the MDH of strain SolV was identified to be a xoxF-ortholog, phylogenetically closely related to mxaF. Anal. of the protein structure and alignment of amino acids showed potential REE-binding motifs in XoxF enzymes of many methylotrophs, suggesting that these may also be lanthanide-dependent MDHs. The authors' findings will have major environmental implications as metagenome studies showed (lanthanide-contg.) XoxF-type MDH is much more prominent in nature than MxaF-type enzymes.
3. 3

   Cotruvo, J. A.; Featherston, E. R.; Mattocks, J. A.; Ho, J. V.; Laremore, T. N. Lanmodulin: A Highly Selective Lanthanide-Binding Protein from a Lanthanide-Utilizing Bacterium. J. Am. Chem. Soc. 2018, 140 (44), 15056– 15061,  DOI: 10.1021/jacs.8b09842

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   Lanmodulin: A Highly Selective Lanthanide-Binding Protein from a Lanthanide-Utilizing Bacterium

   Cotruvo, Joseph A.; Featherston, Emily R.; Mattocks, Joseph A.; Ho, Jackson V.; Laremore, Tatiana N.

   Journal of the American Chemical Society (2018), 140 (44), 15056-15061CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

   Lanthanides (Lns) have been shown recently to be essential cofactors for certain enzymes in methylotrophic bacteria. Here, we identified in the model methylotroph, Methylobacterium extorquens, a highly selective Ln(III)-binding protein, which we named lanmodulin (LanM). LanM possesses 4 metal-binding EF-hand motifs, commonly assocd. with Ca(II)-binding proteins. In contrast to other EF hand-contg. proteins, however, LanM undergoes a large conformational change from a largely disordered state to a compact, ordered state in response to picomolar concns. of all Ln(III) (Ln = La-Lu, Y), whereas it only responded to Ca(II) at near-millimolar concns. Mutagenesis of conserved Pro residues present in LanM's EF hands, not encountered in Ca(II)-binding EF hands, to Ala residues pushed Ca(II) responsiveness into the micromolar concn. range while retaining picomolar Ln(III) affinity, suggesting that these unique Pro residues play a key role in ensuring metal selectivity in vivo. Identification and characterization of LanM provided insights into how biol. selectively recognizes low-abundance Ln(III) over higher-abundance Ca(II), pointing toward biotechnologies for detecting, sequestering, and sepg. these technol. important elements.
4. 4

   Mattocks, J. A.; Jung, J. J.; Lin, C.-Y.; Dong, Z.; Yennawar, N. H.; Featherston, E. R.; Kang-Yun, C. S.; Hamilton, T. A.; Park, D. M.; Boal, A. K.; Cotruvo, J. A. Enhanced Rare-Earth Separation with a Metal-Sensitive Lanmodulin Dimer. Nature 2023, 618 (7963), 87– 93,  DOI: 10.1038/s41586-023-05945-5

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   Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer

   Mattocks, Joseph A.; Jung, Jonathan J.; Lin, Chi-Yun; Dong, Ziye; Yennawar, Neela H.; Featherston, Emily R.; Kang-Yun, Christina S.; Hamilton, Timothy A.; Park, Dan M.; Boal, Amie K.; Cotruvo Jr, Joseph A.

   Nature (London, United Kingdom) (2023), 618 (7963), 87-93CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)

   Technol. crit. rare-earth elements are notoriously difficult to sep., owing to their subtle differences in ionic radius and coordination no.1-3. The natural lanthanide-binding protein lanmodulin (LanM)4,5 is a sustainable alternative to conventional solvent-extn.-based sepn.6. Here we characterize a new LanM, from Hansschlegelia quercus (Hans-LanM), with an oligomeric state sensitive to rare-earth ionic radius, the lanthanum(III)-induced dimer being >100-fold tighter than the dysprosium(III)-induced dimer. X-ray crystal structures illustrate how picometre-scale differences in radius between lanthanum(III) and dysprosium(III) are propagated to Hans-LanMs quaternary structure through a carboxylate shift that rearranges a second-sphere hydrogen-bonding network. Comparison to the prototypal LanM from Methylorubrum extorquens reveals distinct metal coordination strategies, rationalizing Hans-LanMs greater selectivity within the rare-earth elements. Finally, structure-guided mutagenesis of a key residue at the Hans-LanM dimer interface modulates dimerization in soln. and enables single-stage, column-based sepn. of a neodymium(III)/dysprosium(III) mixt. to >98% individual element purities. This work showcases the natural diversity of selective lanthanide recognition motifs, and it reveals rare-earth-sensitive dimerization as a biol. principle by which to tune the performance of biomol.-based sepn. processes.
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   Nitz, M.; Franz, K. J.; Maglathlin, R. L.; Imperiali, B. A Powerful Combinatorial Screen to Identify High-Affinity Terbium(III)-Binding Peptides. ChemBioChem 2003, 4 (4), 272– 276,  DOI: 10.1002/cbic.200390047

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   A powerful combinatorial screen to identify high-affinity terbium(III)-binding peptides

   Nitz, Mark; Franz, Katherine J.; Maglathlin, Rebecca L.; Imperiali, Barbara

   ChemBioChem (2003), 4 (4), 272-276CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)

   Lanthanide-binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodol. for the identification of new LBT sequences with high affinity for Tb3+ ions and intense luminescence properties. This methodol. utilizes solid-phase split-and-pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two-step screening procedure. The initial screen avoids the interference caused by on-bead screening by photochem. releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aq. soln. Employment of this methodol. on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140-fold increase in affinity (57 nM dissocn. const., KD) over previously reported lanthanide-binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2-nM apparent dissocn. const. for Tb3+ ions.
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   Featherston, E. R.; Issertell, E. J.; Cotruvo, J. A., Jr Probing Lanmodulin’s Lanthanide Recognition via Sensitized Luminescence Yields a Platform for Quantification of Terbium in Acid Mine Drainage. J. Am. Chem. Soc. 2021, 143 (35), 14287– 14299,  DOI: 10.1021/jacs.1c06360

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   Probing Lanmodulin's Lanthanide Recognition via Sensitized Luminescence Yields a Platform for Quantification of Terbium in Acid Mine Drainage

   Featherston, Emily R.; Issertell, Edward J.; Cotruvo Jr., Joseph A.

   Journal of the American Chemical Society (2021), 143 (35), 14287-14299CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

   Lanmodulin is the first natural, selective macrochelator for f elements-a protein that binds lanthanides with picomolar affinity at 3 EF hands, motifs that instead bind calcium in most other proteins. Here, we use sensitized terbium luminescence to probe the mechanism of lanthanide recognition by this protein as well as to develop a terbium-specific biosensor that can be applied directly in environmental samples. By incorporating tryptophan residues into specific EF hands, we infer the order of metal binding of these three sites. Despite lanmodulin's remarkable lanthanide binding properties, its coordination of approx. two solvent mols. per site (by luminescence lifetime) and metal dissocn. kinetics (koff = 0.02-0.05 s-1, by stopped-flow fluorescence) are revealed to be rather ordinary among EF hands; what sets lanmodulin apart is that metal assocn. is nearly diffusion limited (kon ∼109 M-1 s-1). Finally, we show that Trp-substituted lanmodulin can quantify 3 ppb (18 nM) terbium directly in acid mine drainage at pH 3.2 in the presence of a 100-fold excess of other rare earths and a 100 000-fold excess of other metals using a plate reader. These studies not only yield insight into lanmodulin's mechanism of lanthanide recognition and the structures of its metal binding sites but also show that this protein's unique combination of affinity and selectivity outperforms synthetic luminescence-based sensors, opening the door to rapid and inexpensive methods for selective sensing of individual lanthanides in the environment and in-line monitoring in industrial operations.
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   Mattocks, J. A.; Ho, J. V.; Cotruvo, J. A. A Selective, Protein-Based Fluorescent Sensor with Picomolar Affinity for Rare Earth Elements. J. Am. Chem. Soc. 2019, 141 (7), 2857– 2861,  DOI: 10.1021/jacs.8b12155

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   A Selective, Protein-Based Fluorescent Sensor with Picomolar Affinity for Rare Earth Elements

   Mattocks, Joseph A.; Ho, Jackson V.; Cotruvo, Joseph A., Jr.

   Journal of the American Chemical Society (2019), 141 (7), 2857-2861CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

   Sensitive yet rapid methods for detection of rare earth elements (REEs), including lanthanides (Lns), would facilitate mining and recycling of these elements. Here we report a highly selective, genetically encoded fluorescent sensor for Lns, LaMP1, based on the recently characterized protein, lanmodulin. LaMP1 displays a 7-fold ratiometric response to all LnIIIs, with apparent Kds of 10-50 pM but only weak response to other common divalent and trivalent metal ions. We use LaMP1 to demonstrate for the first time that a Ln-utilizing bacterium, Methylobacterium extorquens, selectively transports early Lns (LaIII-NdIII) into its cytosol, a surprising observation as the only Ln-proteins identified to date are periplasmic. Finally, we apply LaMP1 to suggest the existence of a LnIII uptake system utilizing a secreted metal chelator, akin to siderophore-mediated FeIII acquisition. LaMP1 not only sheds light on Ln biol. but also may be a useful technol. for detecting and quantifying REEs in environmental and industrial samples.
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   Akerboom, J.; Rivera, J. D. V.; Guilbe, M. M. R.; Malavé, E. C. A.; Hernandez, H. H.; Tian, L.; Hires, S. A.; Marvin, J. S.; Looger, L. L.; Schreiter, E. R. Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design. J. Biol. Chem. 2009, 284 (10), 6455– 6464,  DOI: 10.1074/jbc.M807657200

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   Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

   Akerboom, Jasper; Velez Rivera, Jonathan D.; Rodriguez Guilbe, Maria M.; Alfaro Malave, Elisa C.; Hernandez, Hector H.; Tian, Lin; Hires, S. Andrew; Marvin, Jonathan S.; Looger, Loren L.; Schreiter, Eric R.

   Journal of Biological Chemistry (2009), 284 (10), 6455-6464CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)

   The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as soln. biophys. and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concns. obsd. during imaging expts. and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca2+-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chem. environment of the circularly permuted enhanced green fluorescent protein chromophore and together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.
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   Gifford, J. L.; Walsh, M. P.; Vogel, H. J. Structures and Metal-Ion-Binding Properties of the Ca²⁺-Binding Helix-Loop-Helix EF-Hand Motifs. Biochem. J. 2007, 405 (2), 199– 221,  DOI: 10.1042/BJ20070255

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   Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs

   Gifford, Jessica L.; Walsh, Michael P.; Vogel, Hans J.

   Biochemical Journal (2007), 405 (2), 199-221CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)

   A review. The 'EF-hand' Ca2+-binding motif plays an essential role in eukaryotic cellular signaling, and the proteins contg. this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix-loop-helix secondary structure as well as the ligands presented by the loop to bind the Ca2+ ion. The identity of these ligands is semi-conserved in the most common (the 'canonical') EF-hand; however, several non-canonical EF-hands exist that bind Ca2+ by a different co-ordination mechanism. EF-hands tend to occur in pairs, which form a discrete domain so that most family members have two, four or six EF-hands. This pairing also enables communication, and many EF-hands display pos. co-operativity, thereby minimizing the Ca2+ signal required to reach protein satn. The conformational effects of Ca2+ binding are varied, function-dependent and, in some cases, minimal, but can lead to the creation of a protein target interaction site or structure formation from a molten-globule apo state. EF-hand proteins exhibit various sensitivities to Ca2+, reflecting the intrinsic binding ability of the EF-hand as well as the degree of co-operativity in Ca2+ binding to paired EF-hands. Two addnl. factors can influence the ability of an EF-hand to bind Ca2+: Selectivity over Mg2+ (a cation with very similar chem. properties to Ca2+ and with a cytoplasmic concn. several orders of magnitude higher) and interaction with a protein target. A structural approach is used in this review to examine the diversity of family members, and a biophys. perspective provides insight into the ability of the EF-hand motif to bind Ca2+ with a wide range of affinities.
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    Wang, Q.; Shui, B.; Kotlikoff, M. I.; Sondermann, H. Structural Basis for Calcium Sensing by GCaMP2. Struct. London Engl. 1993 2008, 16 (12), 1817– 1827,  DOI: 10.1016/j.str.2008.10.008

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    Structural basis for calcium sensing by GCaMP2

    Wang Qi; Shui Bo; Kotlikoff Michael I; Sondermann Holger

    Structure (London, England : 1993) (2008), 16 (12), 1817-27 ISSN:0969-2126.

    Genetically encoded Ca(2+) indicators are important tools that enable the measurement of Ca(2+) dynamics in a physiologically relevant context. GCaMP2, one of the most robust indicators, is a circularly permutated EGFP (cpEGFP)/M13/calmodulin (CaM) fusion protein that has been successfully used for studying Ca(2+) fluxes in vivo in the heart and vasculature of transgenic mice. Here we describe crystal structures of bright and dim states of GCaMP2 that reveal a sophisticated molecular mechanism for Ca(2+) sensing. In the bright state, CaM stabilizes the fluorophore in an ionized state similar to that observed in EGFP. Mutational analysis confirmed critical interactions between the fluorophore and elements of the fused peptides. Solution scattering studies indicate that the Ca(2+)-free form of GCaMP2 is a compact, predocked state, suggesting a molecular basis for the relatively rapid signaling kinetics reported for this indicator. These studies provide a structural basis for the rational design of improved Ca(2+)-sensitive probes.
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    Cook, E. C.; Featherston, E. R.; Showalter, S. A.; Cotruvo, J. A. Structural Basis for Rare Earth Element Recognition by Methylobacterium Extorquens Lanmodulin. Biochemistry 2019, 58 (2), 120– 125,  DOI: 10.1021/acs.biochem.8b01019

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    Structural basis for rare earth element recognition by Methylobacterium extorquens lanmodulin

    Cook, Erik C.; Featherston, Emily R.; Showalter, Scott A.; Cotruvo, Joseph A.

    Biochemistry (2019), 58 (2), 120-125CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)

    Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally assocd. with CaII binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for LnIIIs and YIII over CaII. Here we present the NMR soln. structure of LanM complexed with YIII. This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-contg. proteins. It also supports the importance of an addnl. carboxylate ligand in contributing to the protein's picomolar affinity for LnIIIs, and it suggests a role of unusual Ni+1-H···Ni hydrogen bonds, in which LanM's unique EF-hand proline residues are engaged, in selective LnIII recognition. This work sets the stage for a detailed mechanistic understanding of LanM's Ln selectivity, which may inspire new strategies for binding, detecting, and sequestering these technol. important metals.
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    Mattocks, J. A.; Tirsch, J. L.; Cotruvo, J. A. Chapter Two - Determination of Affinities of Lanthanide-Binding Proteins Using Chelator-Buffered Titrations. Methods Enzymol. 2021, 651, 23– 61,  DOI: 10.1016/bs.mie.2021.01.044

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    Determination of affinities of lanthanide-binding proteins using chelator-buffered titrations

    Mattocks, Joseph A.; Tirsch, Jonathan L.; Cotruvo, Joseph A., Jr.

    Methods in Enzymology (2021), 651 (Rare-Earth Element Biochemistry: Characterization and Applications of Lanthanide-Binding Biomolecules), 23-61CODEN: MENZAU; ISSN:1557-7988. (Elsevier Inc.)

    The recent discoveries of the first proteins that bind lanthanides as part of their biol. function not only are relevant to the emerging field of lanthanide-dependent biol., but also hold promise to revolutionize the technol. crit. rare earths industry. Although protocols to assess the thermodn. of metal-protein interactions are well established for "traditional" metal ions in biol., the characterization of lanthanide-binding proteins presents a challenge to biochemists due to the lanthanides' Lewis acidity, propensity for hydrolysis, and high-affinity complexes with biol. ligands. These properties necessitate the prepn. of metal stock solns. with very low buffered "free" metal concns. (e.g., femtomolar to nanomolar) for such detns. Herein we describe several protocols to overcome these challenges. First, we present standardization methods for the prepn. of chelator-buffered solns. of lanthanide ions with easily calcd. free metal concns. We also describe how these solns. can be used in concert with anal. methods including UV-visible spectrophotometry, CD spectroscopy, Forster resonance energy transfer (FRET), and sensitized terbium luminescence, in order to accurately det. dissocn. consts. (Kds) of lanthanide-protein complexes. Finally, we highlight how application of these methods to lanthanide-binding proteins, such as lanmodulin, has yielded insights into selective recognition of lanthanides in biol. We anticipate that these protocols will facilitate discovery and characterization of addnl. native lanthanide-binding proteins, will motivate the understanding of their biol. context, and will prompt their applications in biotechnol.
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    Pu, J.; Zinkus-Boltz, J.; Dickinson, B. C. Evolution of a Split RNA Polymerase as a Versatile Biosensor Platform. Nat. Chem. Biol. 2017, 13 (4), 432– 438,  DOI: 10.1038/nchembio.2299

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    Evolution of a split RNA polymerase as a versatile biosensor platform

    Pu, Jinyue; Zinkus-Boltz, Julia; Dickinson, Bryan C.

    Nature Chemical Biology (2017), 13 (4), 432-438CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)

    Biosensors that transduce target chem. and biochem. inputs into genetic outputs are essential for bioengineering and synthetic biol. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein-protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small mols., demonstrating the 'plug-and-play' nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein-protein interactions and trigger RNA nanostructure prodn., protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biol. applications.
14. 14

    Chang, H.-J.; Mayonove, P.; Zavala, A.; De Visch, A.; Minard, P.; Cohen-Gonsaud, M.; Bonnet, J. A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria. ACS Synth. Biol. 2018, 7 (1), 166– 175,  DOI: 10.1021/acssynbio.7b00266

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    A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria

    Chang, Hung-Ju; Mayonove, Pauline; Zavala, Agustin; De Visch, Angelique; Minard, Philippe; Cohen-Gonsaud, Martin; Bonnet, Jerome

    ACS Synthetic Biology (2018), 7 (1), 166-175CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)

    Engineered bacteria promise to revolutionize diagnostics and therapeutics yet many applications are precluded by the limited no. of detectable signals. Here the authors present a general framework to engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-induced dimerization of a single-domain antibody fused to monomeric DNA-Binding Domains (split-DBDs). Using E. coli as a model system, the authors engineer both transmembrane and cytosolic receptors using a VHH for ligand detection and demonstrate the scalability of the platform by using the DBDs of two different transcriptional regulators. The authors provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing interdomain linker regions. Finally, the authors show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of the split-DBD principle and the versatility of antibody-based detection should support the deployment of these receptors into various host to detect ligands for which no receptor is found in nature.
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    Mazé, A.; Benenson, Y. Artificial Signaling in Mammalian Cells Enabled by Prokaryotic Two-Component System. Nat. Chem. Biol. 2020, 16 (2), 179– 187,  DOI: 10.1038/s41589-019-0429-9

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    Artificial signaling in mammalian cells enabled by prokaryotic two-component system

    Maze, Alain; Benenson, Yaakov

    Nature Chemical Biology (2020), 16 (2), 179-187CODEN: NCBABT; ISSN:1552-4450. (Nature Research)

    Augmenting live cells with new signal transduction capabilities is a key objective in genetic engineering and synthetic biol. We showed earlier that two-component signaling pathways could function in mammalian cells, albeit while losing their ligand sensitivity. Here, we show how to transduce small-mol. ligands in a dose-dependent fashion into gene expression in mammalian cells using two-component signaling machinery. First, we engineer mutually complementing truncated mutants of a histidine kinase unable to dimerize and phosphorylate the response regulator. Next, we fuse these mutants to protein domains capable of ligand-induced dimerization, which restores the phosphoryl transfer in a ligand-dependent manner. Cytoplasmic ligands are transduced by facilitating mutant dimerization in the cytoplasm, while extracellular ligands trigger dimerization at the inner side of a plasma membrane. These findings point to the potential of two-component regulatory systems as enabling tools for orthogonal signaling pathways in mammalian cells.
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    Hall, M. P.; Unch, J.; Binkowski, B. F.; Valley, M. P.; Butler, B. L.; Wood, M. G.; Otto, P.; Zimmerman, K.; Vidugiris, G.; Machleidt, T.; Robers, M. B.; Benink, H. A.; Eggers, C. T.; Slater, M. R.; Meisenheimer, P. L.; Klaubert, D. H.; Fan, F.; Encell, L. P.; Wood, K. V. Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate. ACS Chem. Biol. 2012, 7 (11), 1848– 1857,  DOI: 10.1021/cb3002478

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    Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

    Hall, Mary P.; Unch, James; Binkowski, Brock F.; Valley, Michael P.; Butler, Braeden L.; Wood, Monika G.; Otto, Paul; Zimmerman, Kristopher; Vidugiris, Gediminas; Machleidt, Thomas; Robers, Matthew B.; Benink, Helene A.; Eggers, Christopher T.; Slater, Michael R.; Meisenheimer, Poncho L.; Klaubert, Dieter H.; Fan, Frank; Encell, Lance P.; Wood, Keith V.

    ACS Chemical Biology (2012), 7 (11), 1848-1857CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)

    Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochem. and phys. characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high phys. stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degrdn. sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metab. or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.