Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism.

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Weathered granites and soils harbour microbes with lanthanide-dependent methylotrophic enzymes.

BACKGROUND: Prior to soil formation, phosphate liberated by rock weathering is often sequestered into highly insoluble lanthanide phosphate minerals. Dissolution of these minerals releases phosphate and lanthanides to the biosphere. Currently, the microorganisms involved in phosphate mineral dissolution and the role of lanthanides in microbial metabolism are poorly understood. RESULTS: Although there have been many studies of soil microbiology, very little research has investigated microbiomes of weathered rock. Here, we sampled weathered granite and associated soil to identify the zones of lanthanide phosphate mineral solubilisation and genomically define the organisms implicated in lanthanide utilisation. We reconstructed 136 genomes from 11 bacterial phyla and found that gene clusters implicated in lanthanide-based metabolism of methanol (primarily xoxF3 and xoxF5) are surprisingly common in microbial communities in moderately weathered granite. Notably, xoxF3 systems were found in Verrucomicrobia for the first time, and in Acidobacteria, Gemmatimonadetes and Alphaproteobacteria. The xoxF-containing gene clusters are shared by diverse Acidobacteria and Gemmatimonadetes, and include conserved hypothetical proteins and transporters not associated with the few well studied xoxF systems. Given that siderophore-like molecules that strongly bind lanthanides may be required to solubilise lanthanide phosphates, it is notable that candidate metallophore biosynthesis systems were most prevalent in bacteria in moderately weathered rock, especially in Acidobacteria with lanthanide-based systems. CONCLUSIONS: Phosphate mineral dissolution, putative metallophore production and lanthanide utilisation by enzymes involved in methanol oxidation linked to carbonic acid production co-occur in the zone of moderate granite weathering. In combination, these microbial processes likely accelerate the conversion of granitic rock to soil.

Identification of a TonB-Dependent Receptor Involved in Lanthanide Switch by the Characterization of Laboratory-Adapted Methylosinus trichosporium OB3b.

Two methanol dehydrogenases (MDHs), MxaFI and XoxF, have been characterized in methylotrophic and methanotrophic bacteria. MxaFI contains a calcium ion in its active site, whereas XoxF contains a lanthanide ion. Importantly, the expression of MxaFI and XoxF is inversely regulated by lanthanide bioavailability, i.e., the "lanthanide switch." To reveal the genetic and environmental factors affecting the lanthanide switch, we focused on two Methylosinus trichosporium OB3b mutants isolated during routine cultivation. In these mutants, MxaF was constitutively expressed, but lanthanide-dependent XoxF1 was not, even in the presence of 25 µM cerium ions, which is sufficient for XoxF expression in the wild type. Genotyping showed that both mutants harbored a loss-of-function mutation in the CQW49_RS02145 gene, which encodes a TonB-dependent receptor. Gene disruption and complementation experiments demonstrated that CQW49_RS02145 was required for XoxF1 expression in the presence of 25 µM cerium ions. Phylogenetic analysis indicated that CQW49_RS02145 was homologous to the Methylorubrum extorquens AM1 lanthanide transporter gene (lutH). These findings suggest that CQW49_RS02145 is involved in lanthanide uptake across the outer membrane. Furthermore, we demonstrated that supplementation with cerium and glycerol caused severe growth arrest in the wild type. CQW49_RS02145 underwent adaptive laboratory evolution in the presence of cerium and glycerol ions, resulting in a mutation that partially mitigated the growth arrest. This finding implies that loss-of-function mutations in CQW49_RS02145 can be attributed to residual glycerol from the frozen stock. IMPORTANCE Lanthanides are widely used in many industrial applications, including catalysts, magnets, and polishing. Recently, lanthanide-dependent metabolism was characterized in methane-utilizing bacteria. Despite the global demand for lanthanides, few studies have investigated the mechanism of lanthanide uptake by these bacteria. In this study, we identify a lanthanide transporter in Methylosinus trichosporium OB3b and indicate the potential interaction between intracellular lanthanide and glycerol. Understanding the genetic and environmental factors affecting lanthanide uptake should not only help improve the use of lanthanides for the bioconversion of methane into valuable products like methanol but also be of value for developing biomining to extract lanthanides under neutral conditions.

Genetic Modification of Acidithiobacillus ferrooxidans for Rare-Earth Element Recovery under Acidic Conditions.

As global demands for rare-earth elements (REEs) continue to grow, the biological recovery of REEs has been explored as a promising strategy, driven by potential economic and environmental benefits. It is known that calcium-binding domains, including helix-loop-helix EF hands and repeats-in-toxin (RTX) domains, can bind lanthanide ions due to their similar ionic radii and coordination preference to calcium. Recently, the lanmodulin protein from Methylorubrum extorquens was reported, which has evolved a high affinity for lanthanide ions over calcium. Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile, which has been explored for use in bioleaching for metal recovery. In this report, A. ferrooxidans was engineered for the recombinant intracellular expression of lanmodulin. In addition, an RTX domain from the adenylate cyclase protein of Bordetella pertussis, which has previously been shown to bind Tb3+, was expressed periplasmically via fusion with the endogenous rusticyanin protein. The binding of lanthanides (Tb3+, Pr3+, Nd3+, and La3+) was improved by up to 4-fold for cells expressing lanmodulin and 13-fold for cells expressing the RTX domains in both pure and mixed metal solutions. Interestingly, the presence of lanthanides in the growth media enhanced protein expression, likely by influencing protein stability. Both engineered cell lines exhibited higher recoveries and selectivities for four tested lanthanides (Tb3+, Pr3+, Nd3+, and La3+) over non-REEs (Fe2+ and Co2+) in a synthetic magnet leachate, demonstrating the potential of these new strains for future REE reclamation and recycling applications.

Compartment-related aspects of XoxF protein functionality in Methylorubrum extorquens DM4 analysed using its cytoplasmic targeting.

The impact of periplasmic localisation on the functioning of the XoxF protein was evaluated in the well-studied dichloromethane-utilising methylotroph Methylorubrum extorquens DM4, which harbors only one paralogue of the xoxF gene. It was found that the cytoplasmic targeting of XoxF by expression of the corresponding gene without the sequence encoding the N-terminal signal peptide does not impair the activation and lanthanide-dependent regulation of the MxaFI-methanol dehydrogenase genes. Analysis of the viability of ΔxoxF cells complemented with the full-length and truncated xoxF gene also showed that the expression of cytoplasmically targeted XoxF even increases the resistance to acids. These results contradict the proposed function of the XoxF protein as an extracytoplasmic signal sensor. At the same time, the observed dynamics of growth with methanol, as well as with dichloromethane of strains expressing cytoplasmic-targeted XoxF, indicate the probable enzymatic activity of lanthanide-dependent methanol dehydrogenase in this compartment. Herewith, the only available substrate for this enzyme in cells growing with dichloromethane was formaldehyde, which is produced during the primary metabolism of the mentioned halogenated toxicant directly in the cytosol. These findings suggest that the maturation of XoxF-methanol dehydrogenase may occur already in the cytoplasm, while the factors changing affinity of this enzyme for formaldehyde are apparently absent there. Together with the demonstrated functioning of an enhancer-like upstream activating sequence in the promoter region of the xoxF gene in M. extorquens DM4, the obtained information enriches our understanding of the regulation, synthesis and role of the XoxF protein.

Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion.

De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem. We fused two stable proteins with C2 symmetry-a de novo designed dimeric ferredoxin fold and a de novo designed TIM barrel-such that their symmetry axes are aligned to create scaffolds with large cavities that can serve as binding pockets or enzymatic reaction chambers. The crystal structures of two such designs confirm the presence of a 420 cubic Ångström chamber defined by the top of the designed TIM barrel and the bottom of the ferredoxin dimer. We functionalized the scaffold by installing a metal-binding site consisting of four glutamate residues close to the symmetry axis. The protein binds lanthanide ions with very high affinity as demonstrated by tryptophan-enhanced terbium luminescence. This approach can be extended to other metals and cofactors, making this scaffold a modular platform for the design of binding proteins and biocatalysts.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Certain f-block elements-the lanthanides-have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism if they possess the correct size and a +III oxidation state.

Precision Engineering of 2D Protein Layers as Chelating Biogenic Scaffolds for Selective Recovery of Rare-Earth Elements.

Rare-earth elements, which include the lanthanide series, are key components of many clean energy technologies, including wind turbines and photovoltaics. Because most of these 4f metals are at high risk of supply chain disruption, the development of new recovery technologies is necessary to avoid future shortages, which may impact renewable energy production. This paper reports the synthesis of a non-natural biogenic material as a potential platform for bioinspired lanthanide extraction. The biogenic material takes advantage of the atomically precise structure of a 2D crystalline protein lattice with the high lanthanide binding affinity of hydroxypyridinonate chelators. Luminescence titration data demonstrated that the engineered protein layers have affinities for all tested lanthanides in the micromolar-range (dissociation constants) and a higher binding affinity for the lanthanide ions with a smaller ionic radius. Furthermore, competitive titrations confirmed the higher selectivity (up to several orders of magnitude) of the biogenic material for lanthanides compared to other cations commonly found in f-element sources. Lastly, the functionalized protein layers could be reused in several cycles by desorbing the bound metal with citrate solutions. Taken together, these results highlight biogenic materials as promising bioadsorption platforms for the selective binding of lanthanides, with potential applications in the recovery of these critical elements from waste.

Systems analysis of the effect of hydrogen sulfide on the growth of Methylococcus capsulatus Bath.

Methanotrophs are bacteria capable on growing on methane as their sole carbon source. They may provide a promising route for upgrading natural gas into more valuable fuels and chemicals. However, natural gas may contain significant quantities of hydrogen sulfide. Little is known about how hydrogen sulfide affects the growth and physiology of methanotrophs aside from a few studies showing that it is inhibitory. This study investigated how hydrogen sulfide affects the growth and physiology of the model methanotroph, Methylococcus capsulatus Bath. Growth studies demonstrated that hydrogen sulfide inhibits the growth of M. capsulatus Bath when the concentration exceeds 0.5% (v/v). To better understand how hydrogen sulfide is inhibiting the growth of M. capsulatus Bath, transcription and metabolite concentrations were profiled using RNA sequencing and gas chromatography-mass spectrometry, respectively. Our analysis of the differentially expressed genes and changes in metabolite concentrations suggests that hydrogen sulfide inhibits cellular respiration. The cells respond to sulfide stress in part by increasing the rate of sulfide oxidation and by increasing the expression of sulfide quinone reductase and a putative persulfide dioxygenase. In addition, they reduce the expression of the native calcium-dependent methanol dehydrogenase and increase the expression of XoxF, a lanthanide-dependent methanol dehydrogenase. While the reason of this switch in unknown, XoxF has previously been shown to be induced by lanthanides or nitric oxide in methanotrophs. Collectively, these results further our understanding of how methanotrophs respond to sulfide stress and may aid in the engineering of strains resistant to hydrogen sulfide. KEY POINTS: • Hydrogen sulfide inhibits growth of Methylococcus capsulatus Bath • Sulfide stress inhibits cellular respiration • Sulfide stress induces XoxF, a lanthanide-dependent methanol dehydrogenase.

Lanthanide-Dependent Methanol Metabolism of a Proteobacteria-Dominated Community in a Light Lanthanide-Rich Deep Environment.

This study investigated the occurrence and diversity of proteobacterial XoxF-type methanol dehydrogenases (MDHs) in the microbial community that inhabits a fossil organic matter- and sedimentary lanthanide (Ln3+)-rich underground mine environment using a metagenomic and metaproteomic approach. A total of 8 XoxF-encoding genes (XoxF-EGs) and 14 protein sequences matching XoxF were identified. XoxF-type MDHs were produced by Alpha-, Beta-, and Gammaproteobacteria represented by the four orders Methylococcales, Nitrosomonadales, Rhizobiales, and Xanthomonadales. The highest number of XoxF-EG- and XoxF-matching protein sequences were affiliated with Nitrosomonadales and Rhizobiales, respectively. Among the identified XoxF-EGs, two belonged to the XoxF1 clade, five to the XoxF4 clade, and one to the XoxF5 clade, while seven of the identified XoxF proteins belonged to the XoxF1 clade, four to the XoxF4 clade, and three to the XoxF5 clade. Moreover, the accumulation of light lanthanides and the presence of methanol in the microbial mat were confirmed. This study is the first to show the occurrence of XoxF in the metagenome and metaproteome of a deep microbial community colonizing a fossil organic matter- and light lanthanide-rich sedimentary environment. The presented results broaden our knowledge of the ecology of XoxF-producing bacteria as well as of the distribution and diversity of these enzymes in the natural environment.

An engineered azurin with a lanthanide binding site capable of copper sensing.

Proteins with hetero-bimetallic metal centers can catalyze important reactions and are challenging to design. Azurin is a mononuclear copper center that has been extensively studied for electron transfer. Here we inserted the lanthanide binding tag (LBT), which binds lanthanide with sub µM affinity, into the copper binding loop of azurin, while keeping the type 1 copper center unperturbed. The resulting protein, Az-LBT, which has two metal bonding centers, shows strong luminescence upon coordination with Tb3+ and luminescence quenching upon Cu2+ binding. The in vitro luminescence quenching has high metal specificity and a limit-of-detection of 0.65 µM for Cu2+. With the low background from lanthanide's long luminescence lifetime, bacterial cells expressing Az-LBT in the periplasm also shows sensitivity for metal sensing.

Coordination to lanthanide ions distorts binding site conformation in calmodulin.

The Ca2+-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Ca2+ exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca2+ and several trivalent lanthanide ions) and the carboxylate groups in CaM's EF-hand ion-coordinating sites. Since Tb3+ is commonly used as a luminescent Ca2+ analog in studies of protein-ion binding, it is important to characterize distinctions between the coordination of Ca2+ and the lanthanides in CaM. Although functional assays indicate that Tb3+ fully activates many Ca2+-dependent proteins, our FTIR spectra indicate that Tb3+, La3+, and Lu3+ disrupt the bidentate coordination geometry characteristic of the CaM binding sites' strongly conserved position 12 glutamate residue. The 2D IR spectra indicate that, relative to the Ca2+-bound form, lanthanide-bound CaM exhibits greater conformational flexibility and larger structural fluctuations within its binding sites. Time-dependent 2D IR lineshapes indicate that binding sites in Ca2+-CaM occupy well-defined configurations, whereas binding sites in lanthanide-bound-CaM are more disordered. Overall, the results show that binding to lanthanide ions significantly alters the conformation and dynamics of CaM's binding sites.

E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein.

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

Location-Dependent Lanthanide Selectivity Engineered into Structurally Characterized Designed Coiled Coils.

Herein we report unprecedented location-dependent, size-selective binding to designed lanthanide (Ln3+ ) sites within miniature protein coiled coil scaffolds. Not only do these engineered sites display unusual Ln3+ selectivity for moderately large Ln3+ ions (Nd to Tb), for the first time we demonstrate that selectivity can be location-dependent and can be programmed into the sequence. A 1 nm linear translation of the binding site towards the N-terminus can convert a selective site into a highly promiscuous one. An X-ray crystal structure, the first of a lanthanide binding site within a coiled coil to be reported, coupled with CD studies, reveal the existence of an optimal radius that likely stems from the structural constraints of the coiled coil scaffold. To the best of our knowledge this is the first report of location-dependent metal selectivity within a coiled coil scaffold, as well as the first report of location-dependent Ln3+ selectivity within a protein.

Lanthanide-Binding Tags for 3D X-ray Imaging of Proteins in Cells at Nanoscale Resolution.

We report the application of lanthanide-binding tags (LBTs) for two- and three-dimensional X-ray imaging of individual proteins in cells with a sub-15 nm beam. The method combines encoded LBTs, which are tags of minimal size (ca. 15-20 amino acids) affording high-affinity lanthanide ion binding, and X-ray fluorescence microscopy (XFM). This approach enables visualization of LBT-tagged proteins while simultaneously measuring the elemental distribution in cells at a spatial resolution necessary for visualizing cell membranes and eukaryotic subcellular organelles.

Characterizing the general chelating affinity of serum protein fetuin for lanthanides.

Fetuin is an abundant blood protein that participates in multiple biological processes, including the transport and regulation of calcium. Fetuin is also known to have a high affinity for uranium (as the uranyl dioxo cation) and plutonium, thus it has been suggested as one of the main endogenous chelating biomolecules involved in the transport of actinides following an internal uptake event. Nevertheless, no direct measurements of its affinity for f-elements beside these two actinides have been reported. Here, we investigate the interaction between fetuin and trivalent lanthanides, such as samarium, europium, terbium, and dysprosium, by mass spectrometry and fluorescence spectroscopy. Mass spectrometry results indicated that fetuin has four metal binding sites for the metal ions studied. Upon formation, the metal-protein complexes showed luminescence emission as a result of antenna sensitization of the metal ions, whose photophysics were characterized and exploited to perform direct spectrofluorimetric titrations. Furthermore, the thermodynamic constants were calculated for all complexes, confirming the formation of stable complexes with log [Formula: see text] values between 26 and 27. In characterizing the affinity of the serum protein fetuin for several f-elements, this study expands upon the initial findings focused on uranyl and plutonium, and contributes to a better understanding of the internal distribution and deposition of lanthanides, potentially representative of trivalent actinides.

New Microporous Lanthanide Organic Frameworks. Synthesis, Structure, Luminescence, Sorption, and Catalytic Acylation of 2-Naphthol.

New metal-organic frameworks (MOF) with lanthanum(III), cerium(III), neodymium(III), europium(III), gadolinium(III), dysprosium(III), and holmium(III)] and the ligand precursor 1,3,5-tris(4-carboxyphenyl)-2,4,6-trimethylbenzene (H3L) were synthesized under solvothermal conditions. Single crystal x-ray analysis confirmed the formation of three-dimensional frameworks of [LnL(H2O)2]n·xDMF·yH2O for Ln = La, Ce, and Nd. From the nitrogen sorption experiments, the compounds showed permanent porosity with Brunauer-Emmett-Teller (BET) surface areas of about 400 m2/g, and thermal stability up to 500 °C. Further investigations showed that these Ln-MOFs exhibit catalytic activity, paving the way for potential applications within the field of catalysis.

A Mutagenic Screen Identifies a TonB-Dependent Receptor Required for the Lanthanide Metal Switch in the Type I Methanotroph "Methylotuvimicrobium buryatense" 5GB1C.

Several of the metabolic enzymes in methanotrophic bacteria rely on metals for both their expression and their catalysis. The MxaFI methanol dehydrogenase enzyme complex uses calcium as a cofactor to oxidize methanol, while the alternative methanol dehydrogenase XoxF uses lanthanide metals such as lanthanum and cerium for the same function. Lanthanide metals, abundant in the earth's crust, strongly repress the transcription of mxaF yet activate the transcription of xoxF This regulatory program, called the "lanthanide switch," is central to methylotrophic metabolism, but only some of its components are known. To uncover additional components of the lanthanide switch, we developed a chemical mutagenesis system in the type I gammaproteobacterial methanotroph "Methylotuvimicrobium buryatense" 5GB1C and designed a selection system for mutants unable to repress the mxaF promoter in the presence of lanthanum. Whole-genome resequencing for multiple lanthanide switch mutants identified several unique point mutations in a single gene encoding a TonB-dependent receptor, which we have named LanA. The LanA TonB-dependent receptor is absolutely required for the lanthanide switch and controls the expression of a small set of genes. While mutation of the lanA gene does not affect the amount of cell-associated lanthanum, it is essential for growth in the absence of the MxaF methanol dehydrogenase, suggesting that LanA is involved in lanthanum uptake to supply the XoxF methanol dehydrogenase with its critical metal ion cofactor. The discovery of this novel component of the lanthanide regulatory system highlights the complexity of this circuit and suggests that further components are likely involved.IMPORTANCE Lanthanide metals, or rare earth elements, are abundant in nature and used heavily in technological devices. Biological interactions with lanthanides are just beginning to be unraveled. Until very recently, microbial mechanisms of lanthanide metal interaction and uptake were unknown. The TonB-dependent receptor LanA is the first lanthanum receptor identified in a methanotroph. Sequence homology searches with known metal transporters and regulators could not be used to identify LanA or other lanthanide metal switch components, and this method for mutagenesis and selection was required to identify the receptor. This work advances the knowledge of microbe-metal interactions in environmental niches that impact atmospheric methane levels and are thus relevant to climate change.

Structural Basis for Rare Earth Element Recognition by Methylobacterium extorquens Lanmodulin.

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 associated 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 nuclear magnetic resonance solution 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-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein's picomolar affinity for LnIIIs, and it suggests a role of unusual N i+1-H···N i 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 technologically important metals.

Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization.

BACKGROUND: Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. METHODS: In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. RESULTS: Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model. CONCLUSIONS: Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.