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

Technologically critical rare-earth elements are notoriously difficult to separate, owing to their subtle differences in ionic radius and coordination number1-3. The natural lanthanide-binding protein lanmodulin (LanM)4,5 is a sustainable alternative to conventional solvent-extraction-based separation6. 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-LanM's 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-LanM's greater selectivity within the rare-earth elements. Finally, structure-guided mutagenesis of a key residue at the Hans-LanM dimer interface modulates dimerization in solution and enables single-stage, column-based separation of a neodymium(III)/dysprosium(III) mixture 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 biological principle by which to tune the performance of biomolecule-based separation processes.

Modulating metal-centered dimerization of a lanthanide chaperone protein for separation of light lanthanides.

Elucidating details of biology's selective uptake and trafficking of rare earth elements, particularly the lanthanides, has the potential to inspire sustainable biomolecular separations of these essential metals for myriad modern technologies. Here, we biochemically and structurally characterize Methylobacterium (Methylorubrum) extorquens LanD, a periplasmic protein from a bacterial gene cluster for lanthanide uptake. This protein provides only four ligands at its surface-exposed lanthanide-binding site, allowing for metal-centered protein dimerization that favors the largest lanthanide, LaIII. However, the monomer prefers NdIII and SmIII, which are disfavored lanthanides for cellular utilization. Structure-guided mutagenesis of a metal-ligand and an outer-sphere residue weakens metal binding to the LanD monomer and enhances dimerization for PrIII and NdIII by 100-fold. Selective dimerization enriches high-value PrIII and NdIII relative to low-value LaIII and CeIII in an all-aqueous process, achieving higher separation factors than lanmodulins and comparable or better separation factors than common industrial extractants. Finally, we show that LanD interacts with lanmodulin (LanM), a previously characterized periplasmic protein that shares LanD's preference for NdIII and SmIII. Our results suggest that LanD's unusual metal-binding site transfers less-desirable lanthanides to LanM to siphon them away from the pathway for cytosolic import. The properties of LanD show how relatively weak chelators can achieve high selectivity, and they form the basis for the design of protein dimers for separation of adjacent lanthanide pairs and other metal ions.

Class I ribonucleotide reductases: metallocofactor assembly and repair in vitro and in vivo.

Incorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an Fe(III)Fe(III)-tyrosyl radical (Y•) cofactor (class Ia), a Mn(III)Mn(III)-Y• cofactor (class Ib), and a Mn(IV)Fe(III) cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our understanding of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields.

A genetically encoded fluorescent sensor for manganese(II), engineered from lanmodulin.

The design of selective metal-binding sites is a challenge in both small-molecule and macromolecular chemistry. Selective recognition of manganese (II)-the first-row transition metal ion that tends to bind with the lowest affinity to ligands, as described by the Irving-Williams series-is particularly difficult. As a result, there is a dearth of chemical biology tools with which to study manganese physiology in live cells, which would advance understanding of photosynthesis, host-pathogen interactions, and neurobiology. Here we report the rational re-engineering of the lanthanide-binding protein, lanmodulin, into genetically encoded fluorescent sensors for MnII, MnLaMP1 and MnLaMP2. These sensors with effective Kd(MnII) of 29 and 7 µM, respectively, defy the Irving-Williams series to selectively detect MnII in vitro and in vivo. We apply both sensors to visualize kinetics of bacterial labile manganese pools. Biophysical studies indicate the importance of coordinated solvent and hydrophobic interactions in the sensors' selectivity. Our results establish lanmodulin as a versatile scaffold for design of selective protein-based biosensors and chelators for metals beyond the f-block.

Electron Paramagnetic Resonance, Electronic Ground State, and Electron Spin Relaxation of Seven Lanthanide Ions Bound to Lanmodulin and the Bioinspired Chelator, 3,4,3-LI(1,2-HOPO).

The electron paramagnetic resonance (EPR) spectra of lanthanide(III) ions besides Gd3+ , bound to small-molecule and protein chelators, are uncharacterized. Here, the EPR properties of 7 lanthanide(III) ions bound to the natural lanthanide-binding protein, lanmodulin (LanM), and the synthetic small-molecule chelator, 3,4,3-LI(1,2-HOPO) ("HOPO"), were systematically investigated. Echo-detected pulsed EPR spectra reveal intense signals from ions for which the normal continuous-wave first-derivative spectra are negligibly different from zero. Spectra of Kramers lanthanide ions Ce3+ , Nd3+ , Sm3+ , Er3+ , and Yb3+ , and non-Kramers Tb3+ and Tm3+ , bound to LanM are more similar to the ions in dilute aqueous:ethanol solution than to those coordinated with HOPO. Lanmodulins from two bacteria, with distinct metal-binding sites, had similar spectra for Tb3+ but different spectra for Nd3+ . Spin echo dephasing rates (1/Tm ) are faster for lanthanides than for most transition metals and limited detection of echoes to temperatures below ~6 to 12 K. Dephasing rates were environment dependent and decreased in the order water:ethanol>LanM>HOPO, which is attributed to decreasing librational motion. These results demonstrate that the EPR spectra and relaxation times of lanthanide(III) ions are sensitive to coordination environment, motivating wider application of these methods for characterization of both small-molecule and biomolecule interactions with lanthanides.

Impact of a Biological Chelator, Lanmodulin, on Minor Actinide Aqueous Speciation and Transport in the Environment.

Minor actinides are major contributors to the long-term radiotoxicity of nuclear fuels and other radioactive wastes. In this context, understanding their interactions with natural chelators and minerals is key to evaluating their transport behavior in the environment. The lanmodulin family of metalloproteins is produced by ubiquitous bacteria and Methylorubrum extorquens lanmodulin (LanM) was recently identified as one of nature's most selective chelators for trivalent f-elements. Herein, we investigated the behavior of neptunium, americium, and curium in the presence of LanM, carbonate ions, and common minerals (calcite, montmorillonite, quartz, and kaolinite). We show that LanM's aqueous complexes with Am(III) and Cm(III) remain stable in carbonate-bicarbonate solutions. Furthermore, the sorption of Am(III) to these minerals is strongly impacted by LanM, while Np(V) sorption is not. With calcite, even a submicromolar concentration of LanM leads to a significant reduction in the Am(III) distribution coefficient (Kd, from >104 to ∼102 mL/g at pH 8.5), rendering it even more mobile than Np(V). Thus, LanM-type chelators can potentially increase the mobility of trivalent actinides and lanthanide fission products under environmentally relevant conditions. Monitoring biological chelators, including metalloproteins, and their biogenerators should therefore be considered during the evaluation of radioactive waste repository sites and the risk assessment of contaminated sites.

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

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 approximately two solvent molecules per site (by luminescence lifetime) and metal dissociation 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 association 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.

Characterization of Americium and Curium Complexes with the Protein Lanmodulin: A Potential Macromolecular Mechanism for Actinide Mobility in the Environment.

Anthropogenic radionuclides, including long-lived heavy actinides such as americium and curium, represent the primary long-term challenge for management of nuclear waste. The potential release of these wastes into the environment necessitates understanding their interactions with biogeochemical compounds present in nature. Here, we characterize the interactions between the heavy actinides, Am3+ and Cm3+, and the natural lanthanide-binding protein, lanmodulin (LanM). LanM is produced abundantly by methylotrophic bacteria, including Methylorubrum extorquens, that are widespread in the environment. We determine the first stability constant for an Am3+-protein complex (Am3LanM) and confirm the results with Cm3LanM, indicating a ∼5-fold higher affinity than that for lanthanides with most similar ionic radius, Nd3+ and Sm3+, and making LanM the strongest known heavy actinide-binding protein. The protein's high selectivity over 243Am's daughter nuclide 239Np enables lab-scale actinide-actinide separations as well as provides insight into potential protein-driven mobilization for these actinides in the environment. The luminescence properties of the Cm3+-LanM complex, and NMR studies of Gd3+-LanM, reveal that lanmodulin-bound f-elements possess two coordinated solvent molecules across a range of metal ionic radii. Finally, we show under a wide range of environmentally relevant conditions that lanmodulin effectively outcompetes desferrioxamine B, a hydroxamate siderophore previously proposed to be important in trivalent actinide mobility. These results suggest that natural lanthanide-binding proteins such as lanmodulin may play important roles in speciation and mobility of actinides in the environment; it also suggests that protein-based biotechnologies may provide a new frontier in actinide remediation, detection, and separations.

Lanthanide-dependent coordination interactions in lanmodulin: a 2D IR and molecular dynamics simulations study.

The biological importance of lanthanides, and the early lanthanides (La3+-Nd3+) in particular, has only recently been recognized, and the structural principles underlying selective binding of lanthanide ions in biology are not yet well established. Lanmodulin (LanM) is a novel protein that displays unprecedented affinity and selectivity for lanthanides over most other metal ions, with an uncommon preference for the early lanthanides. Its utilization of EF-hand motifs to bind lanthanides, rather than the Ca2+ typically recognized by these motifs in other proteins, has led it to be used as a model system to understand selective lanthanide recognition. Two-dimensional infrared (2D IR) spectroscopy combined with molecular dynamics simulations were used to investigate LanM's selectivity mechanisms by characterizing local binding site geometries upon coordination of early and late lanthanides as well as calcium. These studies focused on the protein's uniquely conserved proline residues in the second position of each EF-hand binding loop. We found that these prolines constrain the EF-hands for strong coordination of early lanthanides. Substitution of this proline results in a more flexible binding site to accommodate a larger range of ions but also results in less compact coordination geometries and greater disorder within the binding site. Finally, we identify the conserved glycine in the sixth position of each EF-hand as a mediator of local binding site conformation and global secondary structure. Uncovering fundamental structure-function relationships in LanM informs the development of synthetic biology technologies targeting lanthanides in industrial applications.

Biological, biomolecular, and bio-inspired strategies for detection, extraction, and separations of lanthanides and actinides.

Lanthanides and actinides are elements of ever-increasing technological importance in the modern world. However, the similar chemical and physical properties within these groups make purification of individual elements a challenge. Current industrial standards for the extraction, separation, and purification of these metals from natural sources, recycled materials, and industrial waste are inefficient, relying upon harsh conditions, repetitive steps, and ligands with only modest selectivity. Biological, biomolecular, and bio-inspired strategies towards improving these separations and making them more environmentally sustainable have been researched for many years; however, these methods often have insufficient selectivity for practical application. Recent developments in the understanding of how lanthanides are selectively acquired and used by certain bacteria offer the opportunity for a newer, more efficient take on these designs, as well as the possibility for fundamentally new designs and strategies. Herein, we review current cell-based and biomolecular (primarily small-molecule and protein-based) methods for detection, extraction, and separations of f-block elements. We discuss how the increasing knowledge regarding the selective recognition, uptake, trafficking, and storage of these elements in biological systems has informed and will continue to promote development of novel approaches to achieve these ends.

The czcD (NiCo) Riboswitch Responds to Iron(II).

Iron is essential for nearly every organism, and mismanagement of its intracellular concentrations (either deficiency or excess) contributes to diminished virulence in human pathogens, necessitating intricate metalloregulatory mechanisms. To date, although several metal-responsive riboswitches have been identified in bacteria, none has been shown to respond to FeII. The czcD riboswitch, present in numerous human gut microbiota and pathogens, was recently shown to respond to NiII and CoII but thought not to respond to FeII, on the basis of aerobic, in vitro assays; its function in vivo is not well understood. We constructed a fluorescent sensor using this riboswitch fused to the RNA aptamer, Spinach2. When assayed anaerobically, the resulting sensor responds in vitro to FeII, as well as to MnII, CoII, NiII, and ZnII, but only in the cases of FeII and MnII do the apparent Kd values (0.4 and 11 µM, respectively) fall within the range of labile metal concentrations maintained by known metalloregulators. We also show that the sensor-which is, to the best of our knowledge, the first reversible genetically encoded fluorescent sensor for FeII-responds to iron in Escherichia coli cells. Finally, we demonstrate that the putative metal exporters directly downstream of two czcD riboswitches efficiently rescue iron toxicity in a heterologous expression system. Together, our results indicate that iron merits consideration as a plausible physiological ligand for czcD riboswitches, although a response to general metal stress cannot be ruled out at present.

Selective and Efficient Biomacromolecular Extraction of Rare-Earth Elements using Lanmodulin.

Lanmodulin (LanM) is a recently discovered protein that undergoes a large conformational change in response to rare-earth elements (REEs). Here, we use multiple physicochemical methods to demonstrate that LanM is the most selective macromolecule for REEs characterized to date and even outperforms many synthetic chelators. Moreover, LanM exhibits metal-binding properties and structural stability unseen in most other metalloproteins. LanM retains REE binding down to pH ≈ 2.5, and LanM-REE complexes withstand high temperature (up to 95 °C), repeated acid treatments, and up to molar amounts of competing non-REE metal ions (including Mg, Ca, Zn, and Cu), allowing the protein's use in harsh chemical processes. LanM's unrivaled properties were applied to metal extraction from two distinct REE-containing industrial feedstocks covering a broad range of REE and non-REE concentrations, namely, precombustion coal and electronic waste leachates. After only a single all-aqueous step, quantitative and selective recovery of the REEs from all non-REEs initially present (Li, Na, Mg, Ca, Sr, Al, Si, Mn, Fe, Co, Ni, Cu, Zn, and U) was achieved, demonstrating the universal selectivity of LanM for REEs against non-REEs and its potential application even for industrial low-grade sources, which are currently underutilized. Our work indicates that biosourced macromolecules such as LanM may offer a new paradigm for extractive metallurgy and other applications involving f-elements.

A Periplasmic Binding Protein for Pyrroloquinoline Quinone.

Pyrroloquinoline quinone (PQQ) is an essential redox cofactor in bacterial calcium- and lanthanide-dependent alcohol dehydrogenases. Although certain bacteria are known to synthesize and secrete PQQ, little is known about trafficking of this cofactor within and between cells. Here, we show that a previously uncharacterized periplasmic (solute) binding protein from Methylobacterium extorquens AM1, here renamed PqqT, binds 1 equiv of PQQ with high affinity ( Kd = 50 nM). UV-visible and spectrofluorometric titrations establish that PqqT binds an unhydrated form of PQQ with distinct spectral features from the cofactor in free solution. To our knowledge, PqqT is the first solute-binding protein identified for PQQ and the first protein implicated in cellular trafficking of the cofactor. We propose that PqqT, which is encoded adjacent to a putative ATP-binding cassette transporter in the M. extorquens genome, is involved in uptake of exogenous PQQ to supplement endogenous cofactor biosynthesis. These results support the emerging importance of PQQ transfer within microbial and microbe-host communities.

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.

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

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 biology but also may be a useful technology for detecting and quantifying REEs in environmental and industrial samples.

Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide-Dependent Methanol Dehydrogenase Activity.

Lanthanide (Ln)-dependent methanol dehydrogenases (MDHs) have recently been shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems contain two other proteins, XoxG (a c-type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known. In this work, we have biochemically and structurally characterized these proteins from the methyltroph Methylobacterium extorquens AM1. In contrast to results obtained in an artificial assay system, assays of XoxFs metallated with LaIII , CeIII , and NdIII using their physiological electron acceptor, XoxG, display Ln-independent activities, but the Km for XoxG markedly increases from La to Nd. This result suggests that XoxG's redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The X-ray crystal structure of XoxG provides a structural basis for this reduction potential and insight into the XoxG-XoxF interaction. Finally, the X-ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in the activation of XoxF. These studies enrich our understanding of the underlying chemical principles that enable the activity of XoxF with multiple lanthanides in vitro and in vivo.

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

Lanthanides (Lns) have been shown recently to be essential cofactors in certain enzymes in methylotrophic bacteria. Here we identify in the model methylotroph, Methylobacterium extorquens, a highly selective LnIII-binding protein, which we name lanmodulin (LanM). LanM possesses four metal-binding EF hand motifs, commonly associated with CaII-binding proteins. In contrast to other EF hand-containing proteins, however, LanM undergoes a large conformational change from a largely disordered state to a compact, ordered state in response to picomolar concentrations of all LnIII (Ln = La-Lu, Y), whereas it only responds to CaII at near-millimolar concentrations. Mutagenesis of conserved proline residues present in LanM's EF hands, not encountered in CaII-binding EF hands, to alanine pushes CaII responsiveness into the micromolar concentration range while retaining picomolar LnIII affinity, suggesting that these unique proline residues play a key role in ensuring metal selectivity in vivo. Identification and characterization of LanM provides insights into how biology selectively recognizes low-abundance LnIII over higher-abundance CaII, pointing toward biotechnologies for detecting, sequestering, and separating these technologically important elements.

Copper regulates cyclic-AMP-dependent lipolysis.

Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype.

Recognition- and reactivity-based fluorescent probes for studying transition metal signaling in living systems.

Metals are essential for life, playing critical roles in all aspects of the central dogma of biology (e.g., the transcription and translation of nucleic acids and synthesis of proteins). Redox-inactive alkali, alkaline earth, and transition metals such as sodium, potassium, calcium, and zinc are widely recognized as dynamic signals, whereas redox-active transition metals such as copper and iron are traditionally thought of as sequestered by protein ligands, including as static enzyme cofactors, in part because of their potential to trigger oxidative stress and damage via Fenton chemistry. Metals in biology can be broadly categorized into two pools: static and labile. In the former, proteins and other macromolecules tightly bind metals; in the latter, metals are bound relatively weakly to cellular ligands, including proteins and low molecular weight ligands. Fluorescent probes can be useful tools for studying the roles of transition metals in their labile forms. Probes for imaging transition metal dynamics in living systems must meet several stringent criteria. In addition to exhibiting desirable photophysical properties and biocompatibility, they must be selective and show a fluorescence turn-on response to the metal of interest. To meet this challenge, we have pursued two general strategies for metal detection, termed "recognition" and "reactivity". Our design of transition metal probes makes use of a recognition-based approach for copper and nickel and a reactivity-based approach for cobalt and iron. This Account summarizes progress in our laboratory on both the development and application of fluorescent probes to identify and study the signaling roles of transition metals in biology. In conjunction with complementary methods for direct metal detection and genetic and/or pharmacological manipulations, fluorescent probes for transition metals have helped reveal a number of principles underlying transition metal dynamics. In this Account, we give three recent examples from our laboratory and collaborations in which applications of chemical probes reveal that labile copper contributes to various physiologies. The first example shows that copper is an endogenous regulator of neuronal activity, the second illustrates cellular prioritization of mitochondrial copper homeostasis, and the third identifies the "cuprosome" as a new copper storage compartment in Chlamydomonas reinhardtii green algae. Indeed, recognition- and reactivity-based fluorescent probes have helped to uncover new biological roles for labile transition metals, and the further development of fluorescent probes, including ones with varied Kd values and new reaction triggers and recognition receptors, will continue to reveal exciting and new biological roles for labile transition metals.