Supercharged fluorescent proteins detect lanthanides via direct antennae signaling

Domain: earth_science
Published: 2024-10-24
Editorial outcome: accept

Abstract
Asustainableoperationforharvestingmetalsinthelanthanideseriesisneededtomeettherisingdemandforrareearthelementsacrossdiverseglobalindustries.However,existingmethodsarelimitedintheircapacityfordetectionandcaptureatenvironmentallyandindustriallyrelevantlanthanideconcentrations.Superchargedfluorescentproteinshavesolvent-exposed,negativelychargedresiduesthatpotentiallycreatemultipledirectchelationpocketsforfreelanthanidecations.Here,wedemonstratethatnegativelysuperchargedproteinscanbindandquantitativelyreportconcentrationsoflanthanidesviaanunderutilizedlanthanide-to-chromophorepathwayofenergytransfer.Thetop-performingsensorsdetectlanthanidesinthemicromolartomillimolarrangeandremainunperturbedbyenvironmentallysignificantconcentrationsofcompetingmetals.Asademonstrationoftheversatilityandadaptabilityofthisenergytransfermethod,weshowproximityandsignaltransmissionbetweenthelanthanidesandasupramolecularassemblyofsuperchargedproteins,pavingthewayforthedetectionoflanthanidesviaprogrammableproteinoligomersandmaterials.

Introduction
Rare earth elements (REEs) are critically important to the world economy on account of their unique luminescent, magnetic, and catalytic properties1–3. Comprising the fifteen elements of the lanthanide series, as well as scandium and yttrium, REEs have emerged as non-substitutable elements in a variety of consumer and defense applications, encompassing technologies as diverse as wind turbines, solar cells, high-capacity batteries, image-guided cancer therapies, telecommunications, and night-vision systems4–9.

Biomining presents a sustainable, potentially scalable path toward bolstering global lanthanide reserves via unconventional sources, though the absence of a system with appropriate dynamic range, especially in the environment, poses a significant barrier to practical biosensing and microbial engineering10–13. The lanthanide series elements are naturally luminescent and thus capable of ‘self-sensing’. However, on account of the Laporte selection rule, the radiative 4f–4f orbital transitions are spin-forbidden, and consequently, free lanthanide cations have a diminutive capacity for energy absorption14. To circumvent this photophysical constraint, lanthanide cations can be complexed with organic ligands to sensitize the lanthanide’s intrinsic luminescence via the well-studied antenna effect15–17. In other words, the organic ligand operates as an antenna, transducing energy to an otherwise sub-operative lanthanide beacon. Alternatively, lanthanides can themselves function as donors of energy transfer via overlapping and non-overlapping Förster resonance energy transfer (FRET) to closely positioned fluorescent protein acceptors18. Following the FRET model of energy transfer, molecular proximity gives rise to a highly efficient, distance-dependent energy exchange between donor and acceptor entities. Still, this system requires extensive tempering to bring the lanthanide chelate within the FRET signaling range, thereby limiting its use in vivo or at scale, but highlighting a promising avenue for biosensor development.

An attestation to the supreme adaptability of biology, the lanthanide-binding protein, lanmodulin (LanM), from the methylotrophic bacterium Methylobacterium extorquens has a sensitivity for lanthanides in the picomolar range19. Natively a periplasmic protein with a role in lanthanide utilization, LanM has been repurposed by biotechnologists for nascent applications in the sensing, separation, and sequestration of lanthanides20–22. For instance, the LaMP1 fluorescence-based sensor is a tri-fusion protein composed of a citrine and cyan fluorescent protein separated by the intrinsically disordered LanM domain20. Upon binding to the lanthanide series cations, LanM folds into a tight configuration, bringing the two adjacent fluorescent proteins within signaling range to initiate a measurable FRET. Alternatively, the naturally fluorescent amino acid tryptophan can be harnessed as a molecular antenna to sensitize the luminescence of trivalent terbium, yielding an apparent Kd of 10–100 pM23. Notably, this strategy employs sensitized lanthanide luminescence, instead of FRET; nonetheless, only terbium can be detected, and the energy transfer is inefficient due to tryptophan’s low fluorescence quantum yield24.

Although LanM confers an ultra-high sensitivity to the fluorescence-based detection of lanthanides, its superlative nature could limit its utility as a biosensor in more concentrated environmental streams. Considering that acid mine drainage and other waste streams can contain an upwards of 20–100 µM of total REEs, a sensitivity range congruent with such conditions, whether achieved via LanM or some alternative approach, is needed for practical biosensing applications25–27. Moreover, the functionalization of microbes with the ability to concentrate lanthanides necessitates a platform capable of detecting multiple lanthanides in vivo at concentrations potentially spanning 5–5000 micrograms per kilogram of wet biomass or greater28. Recently, the capture of lanthanides on the surface of cells has yielded new insights, including, inadvertently, evidence that lanthanide cations can readily bind to the negatively charged side chains of aspartate and glutamate, found commonly in the extracellular matrix29. Based on the above, we hypothesized that protein charge engineering could be used to optimally position lanthanide cations for luminescence energy transfer to the internal chromophore of fluorescent proteins.

Here, we demonstrate a simple yet robust strategy for using fluorescent proteins as biosensors for lanthanides. The net charge of proteins can be augmented to a significant degree through extensively mutating the molecular surface30. This process, termed ‘supercharging’, generally preserves the overall structure and function of the protein while opening doors to customizable intermolecular behaviors that can meet specific engineering needs31–34. Hence, we modulate the surface of green fluorescent protein (GFP), yellow fluorescent protein (YFP), and cerulean fluorescent protein (CFP) to enable direct chelation between negatively charged residues and free lanthanide cations. Following an underutilized lanthanide-to-chromophore pathway of energy transfer35, ultraviolet (UV) excitation results in a detectable, dose-dependent signal transmission between trivalent terbium, thulium, dysprosium, and the chromophores of YFP and GFP, but not CFP. Notably, supercharged variants of YFP and GFP respond to lanthanides in the environmentally relevant 10 µM–5 mM range. The robust detection of lanthanides via a ready signal transmission to fluorescent proteins suggests that engineered protein surfaces may provide a green path forward in REE extraction, as well as broad opportunities in the development of biosensors and optically active materials.

Results
Supercharged fluorescent proteins demonstrate energy transfer with lanthanide antennae
Contrary to the established paradigm, in which lanthanides are the acceptors of resonance energy transfer, we hypothesized that lanthanides bound by electrostatic forces to a fluorescent protein surface could be functionalized into antennae for receiving an electromagnetic signal outside the normal excitation range of the internal chromophore (Fig. 1a). Subsequently, the signal could be transmitted via lanthanide-based resonance energy transfer (LRET) to the fluorescent protein for a quantifiable emission in the visible range. The efficiency of LRET, like FRET, is inversely dependent on distance35–37. The energy transfer is measurable by fluorescence microscopy only if the lanthanide and the protein are separated by a nanometer-scale distance (10–100 Å), thereby potentially enabling the detection of lanthanides upon deploying the fluorescent biosensors in various environmental and industrial settings38.

To interface with the lanthanide antennae, we aimed at establishing an expansive biosensor excitation range for a maximal chance of favorable spectral overlap with the emissions of Tb3+, Tm3+, and Dy3+ (Fig. 1b). To that end, we introduced the mutations T65G, V68L, S72A, and T203Y to red-shift and stabilize the series of supercharged GFP variants with net charges of −4, −10, −17, −31, +16, and +33 at pH 7.0, obtaining a series of YFP variants with identical net charges32,39. Likewise, we introduced the mutations Y66W, F146G, N147I, H149D to blue-shift and stabilize the same series of GFP variants, obtaining a series of CFP variants with net charges of −5, −11, −18, −32, +15, and +32 at pH 7.032,40–42. This set of eighteen fluorescent protein biosensors then served as our starting point for selecting the optimal candidates for detecting lanthanides (Fig. 2a, b).

Initially, we measured the fluorescence output of the biosensors in response to excitation in the UV range (250–400 nm). When combined with any of the lanthanides (Tb3+, Tm3+, Dy3+), the negatively charged GFP and YFP variants exhibited a 2–3-fold increase in fluorescence intensity at select excitation wavelengths (Supplementary Fig. 1a and Supplementary Table 1). Similarly, the lanthanides Yb3+, Eu3+, and Sm3+ elicited a roughly 2–3-fold increase in fluorescence intensity when paired with the GFP−10 and YFP−31 variants (Supplementary Table 1). In contrast, the addition of Al3+ produced virtually unchanged excitation spectra at a concentration of 100 µM (Supplementary Fig. 1a), a value set to approximate or even exceed the levels of Al3+ commonly found in acid mine drainage43. Consistent with the Laporte rule, the lanthanides did not measurably luminesce on their own at any excitation wavelength14. The ratio of the fluorescence intensity upon adding the lanthanides to the fluorescence of the biosensors alone appeared to peak in the 340–400 nm excitation range, with 350 nm and 390 nm being the optimal wavelengths for achieving a maximal factor change in response upon exciting the lanthanide antennae bound to the GFP and YFP variants, respectively (Supplementary Fig. 1b). However, given that the wavelengths corresponding to these ratiometric maxima encroach on the excitation ranges of GFP and YFP, we opted to excite our lanthanide-bound system deeper in the UV range, at 340 nm and 360 nm for GFP and YFP, respectively. We rationalized that this approach would minimize crosstalk between FRET donor and acceptor channels while still staying within range for a sufficiently robust ratiometric response37. Interestingly, we observed a peak in the fluorescence output of YFP and GFP at an excitation wavelength of 280 nm, with or without the presence of lanthanides, though the addition of lanthanides enhanced the signal response across all variant conditions (Supplementary Fig. 1a). Hypothetically, the tryptophan residue at position 57, positioned approximately 12–13 Å apart from the chromophore within each of the fluorescent protein variants, could act as an antenna at this excitation wavelength for transferring Förster resonance energy directly to the internal chromophore, or in the presence of lanthanides, to the lanthanide, which in turn transmits the signal to the chromophore for an even greater response (Supplementary Figs. 2–4)44. A byproduct of our use of excitation wavelengths of 340 nm and 360 nm, we excluded the confounding effect of the tryptophan antenna in further analyses.

Importantly, time-resolved fluorescence spectroscopy revealed long-lived luminescence transmission from the lanthanide to the fluorescent protein biosensor across the excitation range of 250–400 nm (Fig. 3a, b), consistent with the intrinsically slow decay of the excited state of lanthanide series elements. In the presence of Tb3+, the GFP−10 and YFP−31 biosensors produced a markedly distinct fluorescence response relative to the protein alone. This difference in response was magnified by time delays of 100 µs and 300 µs between excitation and response detection. Of note, the biosensors alone produced negligible fluorescence when subjected to time-delayed detection, while the Tb3+:biosensor pairs exhibited a comparably modest attenuation in the signal response (Fig. 3, Supplementary Figs. 5 and 6). Thus, the long-lived signal responses of the GFP−10 and YFP−31 biosensors in the presence Tb3+ support the notion that signal detection is dependent on excitation and transmission via the lanthanide antennae.

The excitation ratio, defined as the fluorescence of GFP at its emission maximum (510–520 nm) upon exciting at 340 nm versus 465 nm or the fluorescence of YFP (530–540 nm) upon exciting at 360 nm versus 500 nm, is a measure of LRET efficiency that permits comparisons between lanthanide:biosensor and biosensor-only conditions32. The factor change in excitation ratio, defined as the ratio of the excitation ratio calculated for the lanthanide:biosensor condition versus the biosensor-only condition, permits comparisons across biosensor variants. The addition of Tb3+ to YFP−31 produced the highest factor change in excitation ratio, 1.97±0.25, whereas YFP variants with net charges of −17, −10, and −4 produced factor changes in the excitation ratio of 1.58±0.25, 1.48±0.15, and 1.30±0.17, respectively (Fig. 4a and Supplementary Fig. 7). As expected, the non-interacting positively charged variants of YFP+16 and +33, produced factor changes in excitation ratio of 1.13±0.04 and 1.05±0.03, respectively, indicating decreased binding and detection of lanthanides compared to the wild-type YFP. The addition of Tm3+ and Dy3+ to the YFP series resulted in a similarly stepwise increase in signal as the magnitude of negative surface charge increased, with YFP−31 being the top-performing sensor for each of the lanthanides. The addition of Tb3+ to GFP−10 produced a factor change in excitation ratio of 2.58±0.09, the highest signal among the GFP variants, whereas GFP−31, −17, and −4 produced factor changes in excitation ratio of 1.60±0.06, 2.32±0.25, and 1.82±0.08, respectively, and the non-interacting GFP+16 and +33 produced responses of 1.06±0.24 and 0.93±0.11, respectively (Fig. 4a, Supplementary Fig. 7, Supplementary Fig. 8). Again, the addition of Tm3+ and Dy3+ to the GFP series manifested a similar trend, with the entire series of negatively supercharged GFP variants exhibiting an enhanced signal response relative to the positively supercharged and wild-type sensors (Fig. 4a and Supplementary Fig. 7). Importantly, our top performing sensors, YFP−31 and GFP−10 (Supplementary Fig. 9), demonstrated an approximately 2.5-fold or greater increase in their signal response to Tb3+, Tm3+, and Dy3+ across a dose-dependent 10 µM–5 mM range, consistent with typical ranges of total REEs found in highly polluted streams (Fig. 4b, c). Additional testing with the lanthanides Eu3+, Sm3+, and Yb3+ revealed comparable response ratios and an apparent Kd of 25–30 µM for YFP−31 and 210–500 µM for GFP−10 across all six lanthanide species (Fig. 4b, c and Supplementary Table 1). In summary, our findings suggest that negatively supercharging YFP and GFP enables robust lanthanide binding and antennae signaling via (i) specific and non-specific sensitizing interactions within the electronically manipulable framework of the protein interior and (ii) a near-UV range stimulus resulting in the excitation of lanthanide cations and subsequent energy transfer to the protein chromophore.

Notably, the CFP series was non-performing, irrespective of the net charge (Fig. 4a and Supplementary Fig. 7). The inability of the CFP variants to detect lanthanides could be attributed to the absence of spectral overlap with the radiative energy levels of Tb3+ and Tm3+, as well as the excess overlap with the emission of Dy3+ (Fig. 1b)45. In the absence of overlap, the energy received by the lanthanide antenna cannot be transmitted to the acceptor fluorophore, even if the oppositely charged parties are positioned within range for LRET signaling18. Likewise, excess overlap prevents biosensor readout, but in this case, the antenna excitation outputs a signal that is not readily distinguishable from the signal arising from the biosensor-specific excitation37. Conversely, the excitation ranges of YFP and GFP appear to favorably overlap with potentially radiative configurations of trivalent terbium, thulium, and dysprosium in their excited states (Fig. 1b), thereby enabling the successful transmission of energy from the lanthanide antenna to the fluorescent biosensor.

Supercharged fluorescent protein biosensors do not respond to common interferents
Lanthanide-containing waste streams and deposits, such as mine drainage and electronic scrap, contain a surplus of potentially interfering, soluble metal compounds, including, most commonly, the chloride salts of aluminum and iron43,46–49. A biomining operation would therefore benefit from a system capable of distinguishing between the capture of lanthanides versus non-target metals.

To evaluate the response of our biosensor platform to interfering metals, we measured the fluorescence output of our top performing sensors, GFP−10 and YFP−31, in the presence of Al3+ and Fe2+, first as separate, non-interacting entities (Fig. 5a and Supplementary Fig. 10a), and second, as species competing for binding and detection (Supplementary Fig. 10b). Upon UV excitation, the YFP−31 sensor in combination with 1 mM of Tb3+ exhibited a nearly twofold increase in fluorescence output compared to YFP−31 in combination with 1 mM of non-target Al3+ (Fig. 5a). Notably, the Al3+:YFP−31 condition produced an emission spectrum that was only slightly perturbed compared to the YFP−31-only condition, indicating that the YFP−31 sensor can readily discriminate between Tb3+ and a non-lanthanide, non-luminescent metal species of the same ionic charge (Fig. 5a).

Next, we established a competitive interaction between Tb3+ and Al3+ or Fe2+, holding the signal transmission between 1 mM of Tb3+ and the GFP−10 sensor constant as we introduced 1 nM–1 mM of Al3+ or Fe2+ in separate microplate wells (Supplementary Figs. 10b and 11). As we titrated up the concentration of Al3+, the excitation ratio remained constant, with a scale increase in the concentration of six orders of magnitude producing an unperturbed excitation ratio of 0.013±0.001 (Supplementary Fig. 10b). Likewise, the presence of 10 µM of Fe2+ did not impact LRET signaling between GFP−10 and Tb3+, although concentrations greater than 10 µM resulted in a modest diminution of the signal response. At 1 mM of Fe2+, the signal weakened even more, indicating that an equimolar concentration of Fe2+ in the environment could disrupt the detection of lanthanides (Supplementary Fig. 10b). It should be noted that under the reaction conditions presented herein, Fe2+ is potentially readily oxidized to Fe3+. Irrespective of this detail, iron removal via pH-controllable precipitation presents a viable pre-processing strategy to minimize signal interference prior to biosensor readout47.

Importantly, the GFP−10 and YFP−31 biosensors retained functionality for detecting total lanthanides when subjected to mixed ion solutions resembling actual mining outflows (Fig. 5b–d and Supplementary Fig. 12a, b). First, to isolate any potential inhibitory effects of the contaminant metals, we measured the dose responses of Mg2+, Ca2+, Mn2+, Zn2+, and Cu2+ as single species in a competitive assay with GFP−10:Tb3+ and YFP−31:Tb3+. Apart from moderate perturbations in the fluorescence signal at 1 mM of Zn2+ and 100 µM of Cu2+, the contaminants interfered minimally with Tb3+ detection, even at a 10-fold excess of contaminant relative to the lanthanide (Supplementary Fig. 13a, b). Interestingly, Cu2+ appeared to interfere more significantly with Tb3+ detection by the GFP sensor compared to the YFP sensor (Supplementary Fig. 13). Additionally, our analysis yielded no false positive indications; the contaminants did not elicit biosensor fluorescence on their own (i.e., in the absence of Tb3+) as expected (Supplementary Fig. 14). To examine the GFP−10 and YFP−31 response to a true-to-nature mixed ion solution, we approximated the composition of acid mine drainage found in the Virginia Canyon47. With fidelity to the ratio of contaminants, we subjected GFP−10:Tb3+ and YFP−31:Tb3+ to mixtures of Al3+, Fe2+, Mn2+, Zn2+, and Cu2+, termed ‘VC’, as well as Mn2+, Zn2+, and Cu2+, termed ‘mVC’ (Supplementary Table 2). At an adjusted pH of 7, a 5-fold excess of the VC mixture elicited minimal perturbation of Tb3+ detection (Fig. 5c, d). Of note, an approximately equimolar amount of the mVC mixture produced modest interference with GFP−10 detection of Tb3+ (Fig. 5c), and at an adjusted pH of 5, the contaminant mixtures disrupted the detection of 1 mM of Tb3+ at approximately 10 µM and 1 mM of contaminants for GFP−10:Tb3+ and YFP−31:Tb3+, respectively (Supplementary Fig. 12a, b). In addition to transition metal contaminants, REE-rich process streams often contain a high concentration of the non-luminescent lanthanide lanthanum (La3+)47. Upon measuring the dose response of La3+ in a competitive assay with GFP−10:Tb3+ and YFP−31:Tb3+, we observed no significant perturbation of Tb3+ detection at a 5-fold excess of La3+ relative to Tb3+ (Supplementary Fig. 15a, b).

At concentrations of lanthanides high enough to elicit a strong biosensor response, we hypothesized that aggregates could form due to the interaction of near equimolar amounts of oppositely charged species. To evaluate the effect of potential aggregation on fluorescence signal detection, we incubated mixtures of supercharged GFP variants and 1 mM of Al3+ for 30 minutes prior to fluorescence readout. The Tb3+:GFP pairs exhibited excitation ratios on par with an immediate readout, whereas the Al3+:GFP pairs produced slightly increased excitation ratios compared to the GFP-only conditions. Still, the GFP−10 biosensor, as well as the other GFP variants, can discriminate between Tb3+ and Al3+ following the extended incubation period, highlighting the versatility of our system (Supplementary Fig. 16).

To further validate the functionality of our biosensors in real-world conditions, we assessed other potentially interfering factors, such as ionic strength, temperature, and pH changes (Supplementary Figs. 17–19). Of note, concentrations of NaCl from 0 to 800 mM were found to minimally perturb GFP−10 and YFP−31 detection of 1 mM of Tb3+ (Supplementary Fig. 17). Additionally, the temperature range from 0 °C to 70 °C was conducive to stability in Tb3+:GFP−10 signal transduction while the temperature range from 0 °C to 60 °C was conducive to stability in Tb3+:YFP−31 signal transduction (Supplementary Fig. 18). Finally, the pH dependence of Tb3+ detection was such that below a pH of 5, Tb3+ detection was rendered unlikely and virtually indistinguishable from the absence of Tb3+ (Supplementary Fig. 19).

Higher order structures can also detect lanthanides
Symmetry in protein oligomers gives rise to remarkable complexity in biological systems50–52. Given the ubiquity of multimeric protein assemblies and their capacity for diverse structural and transport functions within living organisms, we set out to create a higher order protein architecture for detecting lanthanides53–56. Using a method termed the ‘supercharged protein assembly’ (SuPrA), we selected two oppositely charged fluorescent proteins from our set of biosensors, CFP+32 and GFP−31, for assembly into a globular CFP+32/GFP−31 protomer (Supplementary Fig. 20)32. After combining equimolar volumes of CFP+32 and GFP−31, we observed a unique FRET interaction on par with the signal manifested in the assembly of a 16-unit CFP+32/GFP−17 protomer with eight-fold symmetry (Fig. 6a)32,57. Upon introducing 1 mM of Tb3+, Tm3+, Dy3+, or Al3+, we disrupted the FRET output of GFP−31 in response to excitation at 433 nm, the optimal excitation wavelength of the donor CFP+32. The pattern of disruption in FRET signaling between CFP+32 and GFP−31, in which each of the lanthanides exhibited a stronger effect than aluminum, implies some combined effect of competitive binding, protomer disassembly, and fluorescence modulation. Notably, upon exciting the protomer system in the UV range, following our protocol for transmitting a signal from a lanthanide antenna to its acceptor fluorophore, the fluorescence modulation appeared to reverse course, with each Ln3+:protomer pair manifesting some degree of signal restoration (Fig. 6b). The UV-excited GFP−31 still responded to the lanthanides with a decrease in fluorescence output, although this time, the disruption in FRET between CFP+32 and GFP−31 appeared to be masking a response potentially arising from signaling between the CFP+32/GFP−31 protomer and the lanthanide antennae. To quantitate this masked signal, we calculated the percentage of GFP−31 fluorescence relative to the unperturbed signal. Dy3+ produced the greatest masked signal, a factor change of 1.01±0.09 in the fluorescence output of UV-excited GFP−31 compared to a factor change of 0.77±0.04 for GFP−31 excited at 433 nm. Similarly, Tm3+ induced a factor change of 0.94±0.05 in the fluorescence of UV-excited GFP−31 compared to a factor change of 0.72±0.11 for GFP−31 excited at 433 nm. Tb3+ appeared to demonstrate a diminutive increase in the UV-excited signal, albeit not significantly (Fig. 6c). Hypothetically, the formation of the supramolecular assembly of fluorescent proteins could have induced a spectral shift toward a less favorable overlap with the radiative transitions of Tb3+ and, in turn, decreased the signal transmission.

Methods
Cloning and purification of supercharged fluorescent proteins
Synthetic genes encoding supercharged variants of YFP, GFP, and CFP were obtained from Twist Bioscience and Integrated DNA Technologies (Supplementary Tables 3−5). Escherichia coli DH10B and BL21 (New England Biolabs) were used for cloning and expression, respectively. Expression plasmids based on the pET system were either constructed through Golden Gate assembly or acquired directly from Twist Bioscience.

Starter cultures for expression were prepared by picking single colonies of E. coli for overnight growth in Luria-Bertani broth at 37 °C and 250 r.p.m. The E. coli were then inoculated into 250 mL of broth and grown at 37 °C and 250 r.p.m. until an OD600 of 0.6 was achieved. The cultures were cooled at 4 °C for 45 minutes before 1 mM isopropyl-β-D-1-thiogalactopyranoside was added to induce protein expression. Following 16 hours of incubation at 18 °C and 250 r.p.m., the cells were harvested by centrifugation (6,000g, 4 °C, 30 minutes). The pellets were then either stored at −20 °C for later use or resuspended in 30 mL of phosphate buffer (2 M NaCl, 20 mM imidazole, 5 mM MgSO4, pH 7.5). Once resuspended, the cells were subjected to a sonication routine for cell lysis, and the supernatant containing the soluble proteins was obtained by centrifugation (35,000g, 4 °C, 30 minutes). The proteins were subsequently purified using HisPur Ni-NTA Resin (Thermo Fisher Scientific) and custom wash (50 mM phosphate buffer, 2 M NaCl, 20 mM imidazole, pH 7.5) and elution buffers (50 mM phosphate buffer, 2 M NaCl, 500 mM imidazole, pH 7.5). Desalting and buffer exchange were performed using Amicon Ultra Centrifugal Filter Units (MilliporeSigma), and the purified proteins were concentrated to a volume of 0.4 mL in 50 mM Tris-HCl (pH 7.0). The concentration of the proteins was measured using the Pierce Coomassie Plus Bradford Assay (Thermo Fisher Scientific), and the purity was confirmed through sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Assaying fluorescence in the presence of lanthanides and interferents
Fluorescence measurements were obtained at room temperature using a Cytation 5 microplate reader (BioTek). The sample volume was 0.1 mL unless otherwise specified, and the experiments were conducted using 96-well clear bottom polystyrene assay plates (Corning). At least three independent measurements were obtained for each of the conditions tested. Solutions of TbCl3, TmCl3, DyCl3, EuCl3, SmCl3, and YbCl3 were prepared by dissolving the compounds in 50 mM Tris-HCl. Likewise, the competing metal salts AlCl3, FeCl2, MgCl2, CaCl2, MnCl2, ZnCl2, and CuCl2 were dissolved in 50 mM Tris-HCl. To minimize aggregation, the proteins were carefully agitated by pipetting up and down before diluting the concentrated stock solutions to 0.1 mg/mL in 50 mM Tris-HCl. The pH was buffered at 7.0 unless otherwise indicated.

Time-resolved luminescence responses of GFP−10 and YFP−31 were measured at 0 µs, 100 µs, and 300 µs in the absence and the presence of 1 mM of Tb3+. The ratio of the protein biosensor’s fluorescence upon excitation at a wavelength outside of its normal excitation range versus at a wavelength within its normal excitation range was obtained as a proxy for the magnitude of the lanthanide-based energy transfer from the lanthanide to the biosensor. This quantity, termed the ‘excitation ratio’, normalizes the signal observed when exciting the lanthanide antenna to the signal observed when exciting the fluorescent protein, thereby enabling comparisons of energy transfer across reaction conditions. Excitation ratios were calculated for GFP−10 and YFP−31 in the presence of 1 nM to 10 mM of Tb3+, Tm3+, Dy3+, Eu3+, Sm3+, and Yb3+. Subsequently, the factor changes in the excitation ratios were calculated to compare the performance of charge variants of GFP, YFP, and CFP in the presence of 1 mM of Tb3+, Tm3+, and Dy3+.

To evaluate luminescence responses to interfering metals, the fluorescence output of GFP−10 and YFP−31 were measured in the presence of Al3+, Fe2+, Mg2+, Ca2+, Mn2+, Zn2+, and Cu2+ via non-competitive and competitive assays. In the non-competitive setup, GFP−10 and YFP−31 responses were assayed in the absence and presence of 1 mM of the competing metal. The response in the presence of 1 mM of Tb3+ served as the positive control. In the competitive setup, the biosensors were incubated with 1 mM of Tb3+ prior to introducing 1 nM–10 mM of the competing metal. To confirm that the results were not dependent on the sequence in which the reagents were added, the biosensors were also incubated with 1 nM–10 mM of the competing metal prior to adding 1 mM of Tb3+. Mixed solutions of Al3+, Fe2+, Mn2+, Zn2+, and Cu2+, as well as Mn2+, Zn2+, and Cu2+, were evaluated via the non-competitive and competitive setup. To assemble the CFP+32/GFP−31 protomer, equimolar volumes of CFP+32 and GFP−31 suspended in 50 mM Tris-HCl were combined and incubated for 10 minutes at room temperature. The fluorescence output of the CFP+32/GFP−31 protomer was measured via the method employed for the individual fluorescent proteins, in the presence of 1 mM of Tb3+, Tm3+, Dy3+ and Al3+.

Statistics and reproducibility
The results are presented as the mean +/− S.D. unless otherwise indicated. No statistical method was used to predetermine sample size, and no data were excluded from the analyses. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Supercharged fluorescent proteins detect lanthanides via direct antennae signalling\nHuang et al.\nGeneral comments\nI found this to be a well written paper and well structured. It has broad interest in that as the authors\nstate, rare earth element recycling is of huge interest and significance, yet there is no good way\n(currently) of measuring the concentrations of lanthanides in solution. I think it is worth publishing.\nSpecific comments\n1. I felt that the paper could go with some sort of table summarizing the key numbers (for example the\ndetection limits and capabilities as well as the ratios of excitation compared to controls. At the moment,\nthe key data that exemplifies the sensor capabilities seems to be a bit buried in the manuscript.\n2. In the Introduction, several concepts could be clarified for the non-specialist, especially given the\nproposed journal in which this would be published. This could be an extra line or two to summarise.\nSpecifically the antenna affect (line 36) and the Forster resonance energy transfer (lines 36 and 39).\nThese are rather crucial for the paper and its methods. These could be summarized more with one or\ntwo lines.\n3. Figure 1 a does not seem to convey very specific or useful information. This could be clearer, or at\nleast re-draw to make the point clearer. See point 2 above – maybe this is partly because for the\nuninitiated, it’s not clear exactly how the antenna excitation works.\n4. line 314. E.coli should be spelt out in full the first time it is used here.\n5. The testing for Al and Fe and Cl interference is excellent. What about very mixed ion solutions with\nother metal contaminants (e.g. Cu, Zn etc), as might be more realistic for real mining streams. I\nwondered if this had been examined?\n6. Did the authiors examine temperature effects on the processes as might be experienced from a waste\nstream (although presumably the actual assay could usually be temperature controlled)?\n7. Citations need to be checked for consistency (some are in capitilised word format, some in sentence\nformat).", "reviewer_2": "In this manuscript, Walker and co-workers describe the use of supercharged fluorescent proteins, which\nhave been developed previously to have highly negative surface charge, to bind the luminescent\nlanthanides Tb, Tm, and Dy. The authors take advantage of a less frequently used approach of having the\nlanthanide emission transfer energy to the fluorescent proteins, altering the emission of the proteins\nand resulting in a ratiometric response to the lanthanides. They also carry out experiments with Al and\nFe to assess selectivity. This is an interesting paper overall, exploiting an indeed underutilized format for\nlanthanide luminescence. I believe the authors should have tested selectivity and potential for\ninterferences more stringently. Because the lanthanide binding sites are generic, some conditions (lower\npH, other competing metals) will likely interfere with sensor response, and the authors need to assess\nthese more completely under conditions more relevant to an actual use case. These suggested\nexperiments are described in more detail below, particularly in reference to p. 12.\nDetailed comments:\nAbstract, line 8: I think the authors should be careful about using the word “novel” to describe the\nlanthanide-to-chromophore mode of antenna signaling. One example of precedent for a Tb chelate as\ndonor and GFP as acceptor, which is the same basic principle used here, is ref. 36, Rajapakse et al., PNAS.\n2010, 107, 13582. This strategy is indeed “underutilized,” as the authors write in line 265; that is more\ncorrect than “novel.”\nLine 29: “the absence of a high-affinity system with appropriate sensitivity for detecting lanthanides both\nin vitro and in vivo poses a significant barrier…” I would suggest that this sentence is not correct. The\nLanM-based sensors (high affinity) have been shown to work in vivo (the LaMP1 sensor) and in vitro\n(TrpLanM, e.g. in acid mine drainage). The authors’ perspective that LanM is too high affinity for certain\napplications is valid, but I would argue that the authors have not shown yet that their sensors have\nsufficiently high affinity and selectivity to work in relevant conditions. Roughly 100 µM affinity at neutral\npH is likely not going to be sufficient below pH 5 or so, and possibly not selective enough to outcompete\nthe much higher concentrations of other metals in typical samples. I would also suggest that it is rather\nunlikely that a sensor with 100 µM affinity will work in cells because of the presence of stronger ligands\nlike phosphate, citrate, etc. See below for additional comments related to this point.\nLine 57 and Discussion: “[LanM-based sensors’] superlative nature limits its utility as biosensor at greater\nthan picomolar scales of lanthanide occupancy in more concentrated environmental streams.” An upper\nlimit of detection will be condition dependent. Under conditions similar to those the authors tested\n(roughly 3 µM protein), the TrpLanM sensor’s upper limit of detection would be 6 µM, not 10 nM\n(Discussion, top of p. 19).\nLine 167: On the point of the CFP series of sensors not performing as well as expected, did the authors\ntry the other visible-emitting lanthanides, Sm and Eu, which might be compatible with one or more of\nthe FPs as well? If so, do they interfere with detection? And relatedly, can the authors comment on\nwhether there might be a way to discriminate between the signals for the various lanthanides that the\nsensors respond to?\nFigure 3 and 4 legends: the authors need to provide more information (e.g., the experimental conditions)\nin these legends. In Figure 4, it is particularly important to provide the protein concentration (0.1\nmg/mL? But micromolar would be more useful than mg/mL), buffer, and pH. In my opinion, nanomolar is\na confusing x-axis unit to use for the binding plots as there is micromolar protein and millimolar metal.\np. 12 and following. I am concerned that the authors did not consider a few things when assessing\nselectivity. The direct competition experiments never look at an excess of the potential interferent,\nwhich is a much more realistic scenario than what the authors test: 1 mM Tb and only up to 1 mM Al and\nFe. Al3+ is not very soluble at pH 7 and Fe2+ will be readily oxidized to insoluble Fe3+. So it is unclear\nhow much of these ions are available to compete against the Tb. It is also important to test ions other\nthan Al3+ and Fe2+. In most sources, there will be far more (10x or greater) other lanthanides like La\nand Ce that would likely compete well with the low amounts of Tb, Dy, and Tm. Tb and Tm are two of the\nrarest lanthanides. Being heavier lanthanides, Tb, Dy, and Tm likely would have somewhat higher affinity\nthan the lighter lanthanides, but perhaps not enough to overcome the much higher concentrations. Also,\nmost sources have high concentrations of other ions that are probably reasonably good competitors for\ngeneric carboxylate sites: Zn, Cu, Ca, Mn, Mg. For example, in ref. 23, there was 20 nM Tb out of the 2\nµM total REEs and >10 mM total interferents like Mg, Mn, and Al, and the pH was 3.2. The authors\nshould test lower pHs than 7 too. I think these experiments are important to demonstrate utility.\nI felt the last section, “Higher order structures can also detect lanthanides,” detracted from the overall\npresentation as it did not seem to be particularly successful. Perhaps it could go in the SI instead.\nOther comments:\nLine 46: LanM is not a transporter. I would perhaps call it instead a periplasmic protein with a role in\nlanthanide usage\np. 3, ref. 22: I suggest citing the primary research paper in ACS Cent Sci that demonstrates rare earth\nseparation, rather than the “News and Views”-type article summarizing that work\nFigure 2B: Do the authors have evidence that lanthanides bind at the specific positions indicated in the\nfigure? If not, the authors may want to clarify that their figure is illustrative of possible interactions.\nThe authors should report the Kds, which seem to be in the 50-500 µM range, in the text.\nWhat was the ionic strength of the buffers for the experiments? It is generally good practice to have at\nleast I = 0.1 for metal titrations to minimize non-specific binding.", "reviewer_3": "The manuscript by Walker and co-workers describes the use of supercharged fluorescent proteins as\nbiosensors that detect lanthanide ions (specifically Tb3+, Tm3+ and Dy3+) in the micromolar to\nmillimolar concentration range. The engineered proteins are suggested as a potential “biomining”\ntechnology for detecting lanthanides in acid mine drainage or other industrial waste streams. A series of\nso-called supercharged GFP analogs, with mutations to increase the number of negatively charged\naspartate and glutamate groups on the surface, were in turn mutated into supercharged CFP and YFP\nmutants. A fluorescent plate reader was used to measure the relative fluorescence emission of the\nprotein when excited at different wavelengths in the presence of varied lanthanide ion concentrations.\nThe results show a statistically significant increase in the “fluorescence excitation ratio” that occurs when\nsupercharged GFP or YFP (but not CFP) are equilibrated in solution with lanthanide ions.\nThe excitation ratio is defined as the observed intensity of a fluorescent protein at its emission maximum\n(e.g., GFP at 510−520 nm) upon exciting in the UV region (250 – 400 nm) relative to the observed\nintensity when excited at the protein’s excitation maximum (e.g., GFP at 465 nm). The presented data\nshow that the excitation ratio increases with lanthanide concentration (up to 1 mM) for GFP and YFP,\nbut not CFP. Moreover, the relative increase in excitation ratio is greater for supercharged proteins than for\nwild-type analogs, with the greatest effect seen for GFP(-10) and YFP(-31) (negative number suffix refers\nto relative surface charge). As a negative control, addition of trivalent Al ions did not significantly alter\nthe excitation ratios, suggesting the observed effects are due to energy transfer between surface bound\nlanthanide ions and the fluorescent protein chromophores. Additional data shows spectral changes\nwhen multimeric FP combinations were equilibrated with lanthanides.\nThe results appear to show a noteworthy perturbation of supercharged fluorescent proteins’ excitation\nspectra that varies with lanthanide ion concentration. However, the data generated by plate reader\nmeasurements does not support any definitive conclusions about the photophysical mechanisms\nresponsible for the observed spectral changes. Additionally, there are several problems with the way the\ndata is presented, the rationalization of the study’s significance, and the manner in which the results are\ndiscussed. Considering these limitations, I cannot recommend publication without\nsignificant additional evidence and revised presentation, as detailed below.\nProblem with the study’s rationale and justification.\nThe manuscript posits that there is a significant need for bio-inspired methods for detecting and\nconcentrating lanthanide metals in industrial waste streams. Certainly, this claim is justifiable, but the\npresentation of prior literature on the subject is confusing and contains unsupported or irrelevant\nassertions.\np. 2 line 28: “Biomining presents a sustainable, potentially scalable path towards bolstering global\nlanthanide reserves via unconventional sources, though the absence of a high-affinity system with\nappropriate sensitivity for detecting lanthanides both in vitro and in vivo poses a significant barrier to\npractical biosensing and microbial engineering.”\nThe concept of biomining is introduced in the 2nd paragraph, which continues to describe\nlanthanide photochemistry and energy transfer processes utilized in lanthanide sensing. This description might fit\nbetter in the 3rd paragraph that discusses prior literature on bio-based lanthanide sensors.\np.2 line 36-38: “Due to the requirement of synthetic chelators and chemical modulation, these systems\nare difficult to utilize in living systems, cheaply or at scale.”\nThis statement is unsupported and doesn’t really seem relevant to the purpose of the study.\nFor one thing, the engineered protein biosensors are supposedly intended for use in industrial waste\nstreams, not living systems. For another, it’s not at all obvious that industrial scale use of engineered\nproteins would be cheaper or more scalable than synthetic chelators.\np. 3 line 59: “Considering that acid mine drainage and other waste streams can contain an upwards of\n20–100 μM of total REEs, a sensitivity range congruent with such conditions is needed for practical\nbiosensing applications.”\nThis statement highlights a need for sensors that detect lanthanides in a concentration range of 10-100\nμM. However, it raises an obvious question as to whether supercharged fluorescent proteins with excess\nsurface asparates and glutamates would remain deprotonated and negatively charged at low pH.\nProblems with data presentation and discussion.\nThe manuscript attempts to show that supercharged fluorescent proteins exhibit altered fluorescence\nexcitation spectra in response to lanthanide binding and that the fluorescence changes are due to\nluminescence resonance energy transfer (LRET) from excited lanthanide ions to protein chromophores.\nHowever, the discussion of LRET is confusing and has errors. Also, the results and discussion are hard to\nfollow.\n- One problem is that the Figure Captions do not adequately describe the data shown in the Figures.\nFigure 3, in particular, should describe the data presented in more detail.\n- The supplementary data includes 8 figures, but only 2 (Supplementary Figs. 2 and 8) appear to be\nreferenced in the main text.\n- Another problem is that the description of LRET is confusing or wrong in certain cases, e.g.,\np. 3 line 52: “the naturally fluorescent amino acid tryptophan can be harnessed as a molecular antenna\nto sensitize the luminescence of trivalent terbium… Notably, this strategy employs a direct LRET, instead\nof FRET\"\nHere, the phenomenon of LRET is confused with chromophore-sensitized, lanthanide luminescence.\nLRET refers to energy transfer from an excited lanthanide ion donor transferring energy non-radiatively to a\nnearby absorbing species (e.g., organic fluorophore) via a dipole-dipole mechanism. Sensitized\nlanthanide luminescence refers to transfer of excitation energy from an aromatic chromophore to a\ncoordinated lanthanide ion via a Dexter energy transfer mechanism (followed by lanthanide\nluminescence).\np. 3 line 90: “Contrary to the established paradigm, in which lanthanides are the acceptors of resonance\nenergy transfer…”\nIf this sentence is referring to LRET, it’s incorrect (see above).\nLimitations of the data.\nThe study relies on plate reader measurements to generate single-wavelength fluorescence emission\ndata at different excitation wavelengths. With this level of analysis, it’s not possible to make any\nmeaningful conclusions about energy transfer processes that underlie the observed changes in excitation\nratios.\nAs far as I can tell, the paper hypothesizes that 1) lanthanides coordinate to negatively charged surface\nresidues on supercharged GFP/YFP; 2) excitation in the near UV (e.g., 340 nm, 360 nm) results in\nexcitation of the lanthanide ions via an unspecified antenna effect; and 3) energy is transferred via LRET\nfrom the lanthanide to the proteins’ chromophore. The paper suggests that a tryptophan residue could\nact as an antenna to populate lanthanides’ excitation levels. This would account for an enhanced\nexcitation ratio at seen at lower-wavelength, UV excitation (e.g., 280 nm, Figure 3a), but it does not\naccount for effects seen when exciting in the near-UV (e.g., 340 nm). The paper suggests (Figure S3) that\nunspecified aromatic residues could act as sensitizers, but these residues don’t absorb in this wavelength\nrange.\nHigher resolution, scanning fluorescence emission and excitation spectroscopy might reveal features that\nshed light on the underlying energy transfer mechanisms. For example, a scanning emission spectrum of\nTb/CFP in the range of 400 to 700 nm might reveal residual Tb emission at wavelengths longer than CFP\nfluorescence (e.g., Tb emission line at 620 nm).\nMore informative would be time-resolved luminescence detection that could reveal a long-lifetime\nemission component that would be direct evidence of sensitized lanthanide emission and LRET.\nThe Cytovita 5 plate reader used in this study should be capable of measuring time resolved luminescence."}, "author_response": "Response to Reviewer #1:\nReviewer #1 (Remarks to the Author):\nSupercharged fluorescent proteins detect lanthanides via direct antennae signaling\nHuang et al.\nGeneral comments\nI found this to be a well written paper and well structured. It has broad interest in that as\nthe authors state, rare earth element recycling is of huge interest and significance, yet\nthere is no good way (currently) of measuring the concentrations of lanthanides in\nsolution. I think it is worth publishing.\nWe thank Reviewer #1 for their insightful and constructive comments, as well as their recognition\nof the broad utility of our work.\nSpecific comments\n1. I felt that the paper could go with some sort of table summarizing the key numbers (for\nexample the detection limits and capabilities as well as the ratios of excitation compared\nto controls. At the moment, the key data that exemplifies the sensor capabilities seems to\nbe a bit buried in the manuscript.\nWe thank the Reviewer for their suggestion to include a summary of pertinent values relating to\nthe capabilities of our sensors. Please find in the Supplemental Information a table summarizing\nthe apparent K values of the GFP–10 and YFP–31 biosensors for free lanthanide cations Tb3+,\nd\nTm3+, Dy3+, Eu3+, Sm3+, and Y3+ (new Supplementary Table 1). These values provide a quantitative\nmeasure of the detection capabilities of the GFP–10 and YFP–31 biosensors.\n2. In the Introduction, several concepts could be clarified for the non-specialist, especially\ngiven the proposed journal in which this would be published. This could be an extra line\nor two to summarise. Specifically the antenna affect (line 36) and the Forster resonance\nenergy transfer (lines 36 and 39). These are rather crucial for the paper and its methods.\nThese could be summarized more with one or two lines.\nFor the non-specialist audience, we have revised the text to add the following about the antenna\neffect:\notherwise defunct lanthanide beacon.\nLikewise, we have included the following sentence about Förster resonance energy transfer:\nLine 40: Following the FRET model of energy transfer, molecular proximity gives rise to a highly\nefficient, distance-dependent energy exchange between donor and acceptor entities.\n3. Figure 1a does not seem to convey very specific or useful information. This could be\nclearer, or at least re-draw to make the point clearer. See point 2 above – maybe this is\npartly because for the uninitiated, it’s not clear exactly how the antenna excitation works.\nWe appreciate the Reviewer’s consideration of the utility of Figure 1a. Indeed, the revisions from\nPoint 2 (i.e., exposition regarding the antenna mechanism and FRET model of energy transfer)\nclarify the purpose of Figure 1a, which is to provide an illustrative description of foundational\nconcepts for a non-specialist audience.\n4. line 314. E.coli should be spelt out in full the first time it is used here.\nWe have revised this line to spell out Escherichia coli.\n5. The testing for Al and Fe and Cl interference is excellent. What about very mixed ion\nsolutions with other metal contaminants (e.g. Cu, Zn etc), as might be more realistic for\nreal mining streams. I wondered if this had been examined?\nWe thank the Reviewer for their suggestion to examine the effect of mixed ion solutions. To\nassess applicability in real mining streams, we first measured the dose responses of each\npotential contaminant as a single species in a competitive assay with GFP–10:Tb3+ and YFP–\n31:Tb3+ to elucidate any potential inhibitory effects. After noticing no issues, we approximated\nthe composition of acid mine drainage found in the Virginia Canyon (Goodman et al., 2023). The\nresultant mixture of Al3+, Fe2+, Mn2+, Zn2+, and Cu2+ (with fidelity to the reported ratio in the\npaper) produced minimal interference in the 1 nM–10 mM range when assayed competitively\n2\nwith 1 mM of Tb3+ and the GFP−10 and YFP−31 biosensors (new Figure 5c). Additionally, the\nGFP−10 and YFP−31 biosensors can readily discriminate between 1 mM of Tb3+ and a 10-fold\nexcess of the Virginia Canyon mixture (new Figure 5b).\n6. Did the authors examine temperature effects on the processes as might be experienced\nfrom a waste stream (although presumably the actual assay could usually be temperature\ncontrolled)?\nWe appreciate the suggestion to examine the effect of temperature on lanthanide−biosensor\nsignal transduction. While the Reviewer is correct in that the assay can be temperature\ncontrolled, we decided to carry out the experiments to further understand the limitations of our\nsystem. Via additional experiments, we determined that the temperature range from 0°C to 70°C\nis conducive to stability in Tb3+:GFP−10 signal transduction while the temperature range from 0°C\nto 60°C is conducive to stability in Tb3+:YFP−31 signal transduction (new Supplementary Figure\n15). These results show that the temperature limitations are linked to the stability of the protein,\nrather than the metal-ion binding.\n7. Citations need to be checked for consistency (some are in capitilised word format, some\nin sentence format).\nWe thank the Reviewer for pointing this out. We have double checked and fixed each of the\ncitations.\nReviewer #2 (Remarks to the Author):\nIn this manuscript, Walker and co-workers describe the use of supercharged fluorescent\nproteins, which have been developed previously to have highly negative surface charge,\nto bind the luminescent lanthanides Tb, Tm, and Dy. The authors take advantage of a less\nfrequently used approach of having the lanthanide emission transfer energy to the\nfluorescent proteins, altering the emission of the proteins and resulting in a ratiometric\nresponse to the lanthanides. They also carry out experiments with Al and Fe to assess\nselectivity. This is an interesting paper overall, exploiting an indeed underutilized format\nfor lanthanide luminescence. I believe the authors should have tested selectivity and\npotential for interferences more stringently. Because the lanthanide binding sites are\ngeneric, some conditions (lower pH, other competing metals) will likely interfere with\nsensor response, and the authors need to assess these more completely under conditions\nmore relevant to an actual use case. These suggested experiments are described in more\ndetail below, particularly in reference to p. 12.\nWe thank Reviewer #2 for their extremely thorough review of our work and suggestions of new\nexperiments, as well as their recognition of the underexplored utility of our method. Below, we\nprovide a comprehensive point-by-point response for your consideration.\nDetailed comments:\nAbstract, line 8: I think the authors should be careful about using the word “novel” to\ndescribe the lanthanide-to-chromophore mode of antenna signaling. One example of\nprecedent for a Tb chelate as donor and GFP as acceptor, which is the same basic principle\nused here, is ref. 36, Rajapakse et al., PNAS. 2010, 107, 13582. This strategy is indeed\n“underutilized,” as the authors write in line 265; that is more correct than “novel.”\nWe thank the Reviewer for calling to attention the distinction between “novel” and\n“underutilized”, and we agree that the latter is more correct. The line in question now reads:\nLine 8: By exploiting an underutilized lanthanide-to-chromophore mode of antennae signaling…\nLine 29: “the absence of a high-affinity system with appropriate sensitivity for detecting\nlanthanides both in vitro and in vivo poses a significant barrier…” I would suggest that\n4\nthis sentence is not correct. The LanM-based sensors (high affinity) have been shown to\nwork in vivo (the LaMP1 sensor) and in vitro (TrpLanM, e.g. in acid mine drainage). The\nauthors’ perspective that LanM is too high affinity for certain applications is valid…\nWe thank the Reviewer for pointing this out. The LanM-based sensors are indeed high affinity,\nand we have modified the line in question to read as follows:\nLine 29: …the absence of a system with appropriate dynamic range for detecting lanthanides,\nespecially in the environment, …\nJust as the Reviewer has stated, we still would like to contend that existing sensors (e.g., LaMP1,\nTrpLanM) may have an affinity that is too high for certain applications (with the addition of Line\n342, as a caveat):\nLine 58: Although LanM confers an ultra-high sensitivity to the fluorescence-based detection of\nlanthanides, its superlative nature could limit its utility as a biosensor…\nLine 342: It should be noted, however, that the upper bound of detection will be condition-\ndependent; the tryptophan-substituted sensor indeed achieves a maximum sensitivity in 1–10 µM\nrange, approaching that of the YFP–31 sensor, when subjected to conditions like those employed\nin this study.\n…but I would argue that the authors have not shown yet that their sensors have\nsufficiently high affinity and selectivity to work in relevant conditions. Roughly 100 µM\naffinity at neutral pH is likely not going to be sufficient below pH 5 or so, and possibly\nnot selective enough to outcompete the much higher concentrations of other metals in\ntypical samples.\nWith respect to the affinity of our sensors in the presence of common interferences and at pH 5,\nplease see our response to the Reviewer’s comment about p. 12 (below and also appended here):\nCa2+, Mn2+, Zn2+, and Cu2+ (new Supplementary Fig. 11, new Supplementary Fig. 12), mixed\ncontaminant metal solutions resembling real-world mine outflows (new Fig. 5b−d, new\nSupplementary Fig. 10), pHs (new Supplementary Fig. 10, new Supplementary Fig. 16), and\ntemperatures (new Supplementary Fig. 15) to reflect relevant conditions for performance,\nselectivity and affinity. Of note, the YFP−31 sensor has an affinity of 25−30 µM at pH 7 (new\nSupplementary Table 1), is selective enough to outcompete at least a 10-fold excess of Mg2+,\nCa2+, Mn2+, Zn2+, and Cu2+ individually (new Supplementary Fig. 11b), is selective enough to\noutcompete at least a 10-fold excess of a “real-world” mine outflow mixture at pH 7 (new Figure\n5d), and is selective enough to outcompete an equimolar amount of a “real-world” mine outflow\nmixture at pH 5 (new Supplementary Fig. 10b). With pre-processing of the outflow mixture,\nincluding filtration and pH treatment, we contend that our sensors, or some iteration of our\nsensor platform, may indeed find a real-world niche in lanthanide sensing.\nI would also suggest that it is rather unlikely that a sensor with 100 µM affinity will work\nin cells because of the presence of stronger ligands like phosphate, citrate, etc. See below\nfor additional comments related to this point.\nThe Reviewer may be correct in positing that our biosensor may not work in cells at natural REE\nlevels. Though we are optimistic that the biosensor may eventually find use in engineered cells\nor synthetic cells, which are capable of transporting or concentrating large amounts of REEs, we\nhave revised our discussion to omit any claims that are not supported by the current literature:\nLine 330: Particularly, in environments where the amount of total lanthanides far exceeds the\nupper bound for detection afforded by previously developed LanM derivatives, such as certain\nacidic discharges and highly concentrated filtration streams…\nLine 57 and Discussion: “[LanM-based sensors’] superlative nature limits its utility as\nbiosensor at greater than picomolar scales of lanthanide occupancy in more concentrated\nenvironmental streams.” An upper limit of detection will be condition dependent. Under\nconditions similar to those the authors tested (roughly 3 µM protein), the TrpLanM\nsensor’s upper limit of detection would be 6 µM, not 10 nM (Discussion, top of p. 19).\nmade the following revisions to soften our claim in Line 58 (formerly Line 57) and expand upon\nthis caveat:\nLine 58: Although LanM confers an ultra-high sensitivity to the fluorescence-based detection of\nlanthanides, its superlative nature could limit its utility as a biosensor…\nLine 342: It should be noted, however, that the upper bound of detection will be condition-\ndependent; the tryptophan-substituted sensor indeed achieves a maximum sensitivity in 1–10 µM\nrange, approaching that of the YFP–31 sensor, when subjected to conditions like those employed\nin this study.\nLine 167: On the point of the CFP series of sensors not performing as well as expected,\ndid the authors try the other visible-emitting lanthanides, Sm and Eu, which might be\ncompatible with one or more of the FPs as well? If so, do they interfere with detection?\nAnd relatedly, can the authors comment on whether there might be a way to discriminate\nbetween the signals for the various lanthanides that the sensors respond to?\nAt the time, we had not tested any other visible-emitting lanthanides. We have now performed\nfurther experiments to add Eu3+, Sm3+, and Y3+ to the list of REEs tested against GFP, YFP (new\nFigure 4b−c), and CFP. Of note, Eu3+, Sm3+, and Y3+ elicited similar dose-responsive ratiometric\nresponses when paired with GFP−10 and YFP−31. However, CFP again showed no response to\nthe additional metals, suggesting that the metals either are not binding or do not provide an\nappropriate energy transfer contingent on orbital overlaps. Since (i) CFP was subjected to the\nsame charge mutations as those introduced in GFP and YFP and (ii) CFP lacks compatibility with\nthe most luminescent energy state of each of the tested REEs, we strongly lean towards this latter\nexplanation:\nof spectral overlap with the radiative energy levels of Tb3+ and Tm3+, as well as the excess overlap\nwith the emission of Dy3+.\nFigure 3 and 4 legends: the authors need to provide more information (e.g., the\nexperimental conditions) in these legends. In Figure 4, it is particularly important to\nprovide the protein concentration (0.1 mg/mL? But micromolar would be more useful\nthan mg/mL), buffer, and pH. In my opinion, nanomolar is a confusing x-axis unit to use\nfor the binding plots as there is micromolar protein and millimolar metal.\nWe thank the Reviewer for their suggestions regarding the readability of our Figures and Figure\nlegends. We have now reported the protein concentration in both mg/mL and µM and included\nbuffer and pH conditions in the figure legends. Additionally, the x-axes in each of the\nconcentration curves are now reported in µM, consistent with the units for protein\nconcentration.\np. 12 and following. I am concerned that the authors did not consider a few things when\nassessing selectivity. The direct competition experiments never look at an excess of the\npotential interferent, which is a much more realistic scenario than what the authors test:\n1 mM Tb and only up to 1 mM Al and Fe. Al3+ is not very soluble at pH 7 and Fe2+ will\nbe readily oxidized to insoluble Fe3+. So it is unclear how much of these ions are\navailable to compete against the Tb. It is also important to test ions other than Al3+ and\nFe2+. In most sources, there will be far more (10x or greater) other lanthanides like La\nand Ce that would likely compete well with the low amounts of Tb, Dy, and Tm. Tb and\nTm are two of the rarest lanthanides. Being heavier lanthanides, Tb, Dy, and Tm likely\nwould have somewhat higher affinity than the lighter lanthanides, but perhaps not enough\nto overcome the much higher concentrations. Also, most sources have high\nconcentrations of other ions that are probably reasonably good competitors for\ngeneric carboxylate sites: Zn, Cu, Ca, Mn, Mg. For example, in ref. 23, there was 20 nM Tb out\nof the 2 µM total REEs and >10 mM total interferents like Mg, Mn, and Al, and the pH\nwas 3.2. The authors should test lower pHs than 7 too. I think these experiments are\nimportant to demonstrate utility.\nWe thank the Reviewer for pointing out the limitations of our competition assays and for their\nsuggestions of additional studies. As recommended, we have performed additional experiments\nwith other contaminant metals, including 10-fold excesses of Mg2+, Ca2+, Mn2+, Zn2+, and Cu2+\n8\n(new Supplementary Fig. 11, new Supplementary Fig. 12), mixed contaminant metal solutions\nresembling real-world mine outflows (new Fig. 5b−d, new Supplementary Fig. 10), pHs (new\nSupplementary Fig. 10, new Supplementary Fig. 16), and temperatures (new Supplementary\nFig. 15) to reflect relevant conditions for performance, selectivity and affinity. Of note, the\nYFP−31 sensor has an affinity of 25−30 µM at pH 7 (new Supplementary Table 1), is selective\nenough to outcompete at least a 10-fold excess of Mg2+, Ca2+, Mn2+, Zn2+, and Cu2+ individually\n(new Supplementary Fig. 11b), is selective enough to outcompete at least a 10-fold excess of a\n“real-world” mine outflow mixture at pH 7 (new Figure 5d), and is selective enough to\noutcompete an equimolar amount of a “real-world” mine outflow mixture at pH 5 (new\nSupplementary Fig. 10b).\nAdditionally, we tested the ability of the supercharged FPs to detect Tb3+ across a range of pH\nvalues (new Supplementary Fig. 16). Evidently, the system performs well above pH 5, potentially\nat pH 4, but not at pH 3. This is likely due to the denaturation of the FPs at low pH. However, in\npractice, we expect to be able to buffer the assay conditions to counteract the lower pH of certain\nsamples. Finally, we tested lighter lanthanides (Eu3+ and Sm3+), as well as Y3+, and found\ncomparable K values across all REEs against GFP−10 and YFP−31 (new Fig. 4b−c, new\nd\nSupplementary Table 1). With pre-processing of the outflow mixture, including filtration and pH\ntreatment, we contend that our sensors, or some computationally guided iteration of our sensor\nplatform, may indeed find a real-world niche in lanthanide sensing.\nMoreover, with respect to the issue of pH in realistic scenarios, we would like to point out that\nin the natural environment, REE streams are sometimes characterized by higher pH values. To\nname one example, the acid mine effluent from the Big 5 Tunnel in Colorado was measured to\nhave a pH of 5.5 (Goodman et al., 2023). As stated above, our analysis indicates that at a pH of 5,\nour biosensors (in particular, YFP−31), still retain a sensitive detection of lanthanides (new\nSupplementary Fig. 16). Importantly as well, work has been done by the U.S. Army ERDC to\nconcentrate REEs from acid mine drainage via a 2-stage pH adjustment process, which also\n9\nremoves Fe and Al from the samples. It is our view that buffering the assay to counteract a low\npH condition is a relatively straightforward task.\nIn most sources, there will be far more (10x or greater) other lanthanides like La and Ce\nthat would likely compete well with the low amounts of Tb, Dy, and Tm. Tb and\nTm are two of the rarest lanthanides.\nTb3+ and Tm3+ are indeed two of the rarest lanthanides. We included them in testing as they have\nsignificant relevance in sonar and laser applications, which are of high interest to our research\ngroup and partners.\nI felt the last section, “Higher order structures can also detect lanthanides,” detracted from\nthe overall presentation as it did not seem to be particularly successful. Perhaps it could\ngo in the SI instead.\nWe thank the Reviewer for their comment. The impact of the experiment is primarily to\ndemonstrate that the approach we have taken is extremely amenable to further engineering\nattempts. In this regard, this experiment shows: (i) the successful demonstration of signal\ntransmission between the lanthanide antennae and the oligomeric protein architecture (Figure\n6b), (ii) the instantiation of higher order programming in terms of lanthanide detection, and (iii)\nthe relevance to engineered materials (via the adaptation of an underutilized sensing paradigm\nto the principles of cross-linking and modular design).\nOther comments:\nLine 46: LanM is not a transporter. I would perhaps call it instead a periplasmic protein\nwith a role in lanthanide usage\nWe thank the Reviewer for noting this; LanM is indeed not a transporter. This line has been\ncorrected and now reads as follows:\nLine 48: Natively a periplasmic protein with a role in lanthanide utilization, LanM…\n10\np. 3, ref. 22: I suggest citing the primary research paper in ACS Cent Sci that demonstrates\nrare earth separation, rather than the “News and Views”-type article summarizing that\nwork\nWe thank the Reviewer for this suggestion. The primary paper is now cited instead:\nDong, Z. et al. Bridging hydrometallurgy and biochemistry: a protein-based process for recovery\nand separation of rare earth elements. ACS Cent. Sci. 7, 1798–1808 (2021).\nFigure 2B: Do the authors have evidence that lanthanides bind at the specific positions\nindicated in the figure? If not, the authors may want to clarify that their figure is\nillustrative of possible interactions.\nThe schematic in Figure 2b is indeed merely an illustration of possible interactions. Thus, we have\nmodified Figure 2b caption to read as follows:\nFigure 2b: An illustration of potential lanthanide binding interactions and proximity to the protein\nchromophore. Solvent-exposed residues are mutated to increase the net charge while preserving\nthe structure and function of the fluorescent protein.\nThe authors should report the Kds, which seem to be in the 50-500 µM range, in the text.\nWe appreciate the Reviewer’s suggestion to include a summary of pertinent values relating to\nthe capabilities of our sensors. Please find in the Supplementary Information a table\nsummarizing the apparent K values of the GFP–10 and YFP–31 biosensors bound to free\nd\nlanthanide cations Tb3+, Tm3+, Dy3+, Eu3+, Sm3+, and Y3+ (new Supplementary Table 1). These\nvalues provide a quantitative measure of the detection capabilities of the GFP–10 and YFP–31\nbiosensors.\nWhat was the ionic strength of the buffers for the experiments? It is generally good\npractice to have at least I = 0.1 for metal titrations to minimize non-specific binding.\nknown that certain buffers are capable of binding REEs. However, we have repeated some of the\nexperiments with different ionic strengths ranging from 0.1 to 0.8 and found that such changes\ndo not affect biosensor performance (new Supplementary Figure 14).\nResponse to Reviewer #3:\nReviewer #3 (Remarks to the Author):\nThe manuscript by Walker and co-workers describes the use of supercharged fluorescent\nproteins as biosensors that detect lanthanide ions (specifically Tb3+, Tm3+ and Dy3+) in\nthe micromolar to millimolar concentration range. The engineered proteins are suggested\nas a potential “biomining” technology for detecting lanthanides in acid mine drainage or\nother industrial waste streams. A series of so-called supercharged GFP analogs, with\nmutations to increase the number of negatively charged aspartate and glutamate groups\non the surface, were in turn mutated into supercharged CFP and YFP mutants. A\nfluorescent plate reader was used to measure the relative fluorescence emission of the\nprotein when excited at different wavelengths in the presence of varied lanthanide ion\nconcentrations. The results show a statistically significant increase in the “fluorescence\nexcitation ratio” that occurs when supercharged GFP or YFP (but not CFP) are\nequilibrated in solution with lanthanide ions.\nThe excitation ratio is defined as the observed intensity of a fluorescent protein at its\nemission maximum (e.g., GFP at 510−520 nm) upon exciting in the UV region (250 –\n400 nm) relative to the observed intensity when excited at the protein’s excitation\nmaximum (e.g., GFP at 465 nm). The presented data show that the excitation ratio\nincreases with lanthanide concentration (up to 1 mM) for GFP and YFP, but not CFP.\nMoreover, the relative increase in excitation ratio is greater for supercharged proteins\nthan for wild-type analogs, with the greatest effect seen for GFP(-10) and YFP(-31)\n(negative number suffix refers to relative surface charge). As a negative control, addition\nof trivalent Al ions did not significantly alter the excitation ratios, suggesting the observed\neffects are due to energy transfer between surface bound lanthanide ions and the\nfluorescent protein chromophores. Additional data shows spectral changes when\nmultimeric FP combinations were equilibrated with lanthanides.\nThe results appear to show a noteworthy perturbation of supercharged fluorescent\nproteins’ excitation spectra that varies with lanthanide ion concentration. However, the\ndata generated by plate reader measurements does not support any definitive conclusions\n12\nabout the photophysical mechanisms responsible for the observed spectral changes.\nAdditionally, there are several problems with the way the data is presented, the\nrationalization of the study’s significance, and the manner in which the results are\ndiscussed. Considering these limitations, I cannot recommend publication without\nsignificant additional evidence and revised presentation, as detailed below.\nWe thank the Reviewer for their constructive comments and thorough review of our study. We\nhave included significant additional evidence, as suggested, which we believe strengthen the\npaper and conclusions to a surprising extent.\nProblem with the study’s rationale and justification.\nThe manuscript posits that there is a significant need for bio-inspired methods for\ndetecting and concentrating lanthanide metals in industrial waste streams. Certainly, this\nclaim is justifiable, but the presentation of prior literature on the subject is confusing and\ncontains unsupported or irrelevant assertions.\nPlease find below a breakdown of our revisions and response.\np. 2 line 28: “Biomining presents a sustainable, potentially scalable path towards\nbolstering global lanthanide reserves via unconventional sources, though the absence of\na high-affinity system with appropriate sensitivity for detecting lanthanides both in vitro\nand in vivo poses a significant barrier to practical biosensing and microbial engineering.”\nThe concept of biomining is introduced in the 2nd paragraph, which continues to describe\nlanthanide photochemistry and energy transfer processes utilized in lanthanide sensing.\nThis description might fit better in the 3rd paragraph that discusses prior literature on bio-\nbased lanthanide sensors.\nWe thank the Reviewer for suggesting this improvement. However, on the contrary, we feel that\nmoving this sentence to the 3rd paragraph would hinder, rather than help, the clarity. The\npurpose of this sentence, in our view, is to introduce a limitation in the current science (i.e., the\nbarrier to practical biosensing). Thus, we believe that it is important to present this concept early\nin the introduction, especially prior to detailing the limitations related to specific lanthanide\ndetection systems, discussed in the first paragraph.\n13\np.2 line 36-38: “Due to the requirement of synthetic chelators and chemical modulation,\nthese systems are difficult to utilize in living systems, cheaply or at scale.”\nThis statement is unsupported and doesn’t really seem relevant to the purpose of the\nstudy. For one thing, the engineered protein biosensors are supposedly intended for use\nin industrial waste streams, not living systems. For another, it’s not at all obvious that\nindustrial scale use of engineered proteins would be cheaper or more scalable than\nsynthetic chelators.\nWe thank the Reviewer for noting this. Indeed, engineered proteins may not be cheaper or more\nscalable than synthetic chelators. Additionally, the Reviewer may be correct in suggesting that\nour biosensor may not work in cells at natural REE levels. Though we are optimistic that the\nbiosensor may eventually find use in engineered cells or synthetic cells, which are capable of\ntransporting or concentrating large amounts of REEs, we have removed the line in question in its\nentirety and revised our discussion to omit any claims about use in living systems that are not\nsupported by the current literature:\nLine 330: Particularly, in environments where the amount of total lanthanides far exceeds the\nupper bound for detection afforded by previously developed LanM derivatives, such as certain\nacidic discharges and highly concentrated filtration streams…\np. 3 line 59: “Considering that acid mine drainage and other waste streams can contain\nan upwards of 20–100 μM of total REEs, a sensitivity range congruent with such\nconditions is needed for practical biosensing applications.”\nThis statement highlights a need for sensors that detect lanthanides in a concentration\nrange of 10-100 μM. However, it raises an obvious question as to whether supercharged\nfluorescent proteins with excess surface asparates and glutamates would remain\ndeprotonated and negatively charged at low pH.\nThe Reviewer raises an excellent point; at ~pH 3.5 and below, there is a strong chance the\naspartates and glutamates would not remain deprotonated. We would like to point out that in\nthe natural environment, REE streams are sometimes characterized by higher pH values. To name\none example, the acid mine effluent from the Big 5 Tunnel in Colorado was measured to have a\n14\npH of 5.5 (Goodman et al., 2023). Our analysis indicates that at a pH of 5, our biosensors (in\nparticular, YFP−31), still retain a sensitive detection of lanthanides (new Supplementary Fig. 16).\nImportantly as well, work has been done by the U.S. Army ERDC to concentrate REEs from acid\nmine drainage via a 2-stage pH adjustment process, which also removes Fe and Al from the\nsamples. It is our view that it is a simple matter to buffer the assay to counteract a low pH\ncondition, in order to accurately measure the REEs in solution.\nProblems with data presentation and discussion.\nThe manuscript attempts to show that supercharged fluorescent proteins exhibit altered\nfluorescence excitation spectra in response to lanthanide binding and that the fluorescence\nchanges are due to luminescence resonance energy transfer (LRET) from excited\nlanthanide ions to protein chromophores. However, the discussion of LRET is confusing\nand has errors. Also, the results and discussion are hard to follow.\nWe thank the Reviewer for these insightful comments. Please find below a breakdown of our\nrevisions and response.\n- One problem is that the Figure Captions do not adequately describe the data shown in\nthe Figures. Figure 3, in particular, should describe the data presented in more detail.\nWe thank the Reviewer for their suggestions regarding the Figure captions. We have now\nincluded more details in each of the Figure captions, both in the main manuscript and the\nsupplemental file. In particular, we have included the protein concentration in both mg/mL and\nµM and included buffer and pH conditions in the figure legends. Additionally, the x-axes in each\nof the concentration curves are now reported in µM, consistent with the units for protein\nconcentration.\n- The supplementary data includes 8 figures, but only 2 (Supplementary Figs. 2 and 8)\nappear to be referenced in the main text.\nWe thank the Reviewer for noting this. We have expanded upon the supplementary data and\nreferenced each of the Figures (Supplementary Figs. 1−17) in the main text.\n15\n- Another problem is that the description of LRET is confusing or wrong in certain cases,\ne.g.,\np. 3 line 52: “the naturally fluorescent amino acid tryptophan can be harnessed as a\nmolecular antenna to sensitize the luminescence of trivalent terbium… Notably, this\nstrategy employs a direct LRET, instead of FRET\"\nHere, the phenomenon of LRET is confused with chromophore-sensitized, lanthanide\nluminescence. LRET refers to energy transfer from an excited lanthanide ion donor\ntransferring energy non-radiatively to a nearby absorbing species (e.g., organic\nfluorophore) via a dipole-dipole mechanism. Sensitized lanthanide luminescence refers\nto transfer of excitation energy from an aromatic chromophore to a coordinated\nlanthanide ion via a Dexter energy transfer mechanism (followed by lanthanide\nluminescence).\nWe thank the Reviewer for their clarification of the relevant mechanisms. Indeed, the line in\nquestion should reference sensitized lanthanide luminescence, rather than LRET. We have now\nrevised the sentence to accurately convey the mechanism of energy transfer:\nLine 56: Notably, this strategy employs sensitized lanthanide luminescence…\np. 3 line 90: “Contrary to the established paradigm, in which lanthanides are the acceptors\nof resonance energy transfer…”\nIf this sentence is referring to LRET, it’s incorrect (see above).\nIn this line, we are indeed referring to the transfer of energy via a Dexter pathway (i.e., the\nmechanism utilized by the TrpLanM sensor from above).\nLimitations of the data.\nThe study relies on plate reader measurements to generate single-wavelength\nfluorescence emission data at different excitation wavelengths. With this level of analysis,\nit’s not possible to make any meaningful conclusions about energy transfer processes that\nunderlie the observed changes in excitation ratios.\nneeded. Below, we detail further experimentation, including time-resolved luminescence\nspectroscopy, to elucidate the mechanism of energy transfer.\nWith respect to single-wavelength emission data, we did perform broad scanning emission\nspectrums (new Fig. 3, Fig. 5a, new Fig. 5b); however, in certain instances we only included single\npeak wavelength emission data to simplify the results, as there was only one emission peak\nwithin the emission scans.\nAs far as I can tell, the paper hypothesizes that 1) lanthanides coordinate to negatively\ncharged surface residues on supercharged GFP/YFP; 2) excitation in the near UV (e.g.,\n340 nm, 360 nm) results in excitation of the lanthanide ions via an unspecified antenna\neffect; and 3) energy is transferred via LRET from the lanthanide to the proteins’\nchromophore. The paper suggests that a tryptophan residue could act as an antenna to\npopulate lanthanides’ excitation levels. This would account for an enhanced excitation\nratio at seen at lower-wavelength, UV excitation (e.g., 280 nm, Figure 3a), but it does not\naccount for effects seen when exciting in the near-UV (e.g., 340 nm). The paper suggests\n(Figure S3) that unspecified aromatic residues could act as sensitizers, but these residues\ndon’t absorb in this wavelength range.\nWe thank the Reviewer for pointing out this inconsistency. While there is a strong excitation at\n280 nm, we have observed an even greater ratiometric response at 340−360 nm when the REEs\nare bound (Supplementary Fig. 1b). We hypothesize that the strong excitation at 280 nm is\naccounted for by the Trp57 antenna (as stated also by the Reviewer). When we excite the system\nat 340−360 nm, outside the range of aromatic residue absorption, it is the lanthanide ion that is\nbeing excited in the absence of an aromatic sensitizer, which in turn transfers energy to the\nchromophore of the biosensors. The unspecified aromatic residues are yet another possible\nexplanation for the strong excitation at or near 280 nm, not 340−360 nm. We have revised the\ncaption of Supplementary Fig. 3 to clarify this point:\nSupplementary Fig. 4: The lanthanide binding sites, aspartate and glutamate, are surrounded by\nan electronically dense network of potentially sensitizing aromatics. In conjunction with Trp57,\nthis unique architecture could account for the signal response upon excitation at or near 280 nm.\nLine 183: In summary, our findings suggest that negatively supercharging YFP and GFP enables\nrobust lanthanide binding and subsequent antennae signaling via (i) specific and non-specific\nsensitizing interactions within the electronically manipulable framework of the protein interior\nand (ii) a near-UV range stimulus resulting in the excitation of lanthanide cations and subsequent\nenergy transfer to the protein chromophore.\nHigher resolution, scanning fluorescence emission and excitation spectroscopy might\nreveal features that shed light on the underlying energy transfer mechanisms. For\nexample, a scanning emission spectrum of Tb/CFP in the range of 400 to 700 nm might\nreveal residual Tb emission at wavelengths longer than CFP fluorescence (e.g., Tb\nemission line at 620 nm).\nWith respect to residual Tb emissions, this is an excellent point; we assumed the same as the\nReviewer. However, we have performed CFP scanning excitation and emission with all of the REEs\n(including the 3 additional REEs (new Fig. 4b−c)) and found no discernable differences in the\npresence or absence of metals.\nMore informative would be time-resolved luminescence detection that could reveal a\nlong-lifetime emission component that would be direct evidence of sensitized lanthanide\nemission and LRET. The Cytovita 5 plate reader used in this study should be capable of\nmeasuring time resolved luminescence.\nWe thank the Reviewer for their suggestion of a more informative experiment (i.e., time-resolved\nluminescence detection). We undertook a series of new time-resolved experiments with 0 μs,\n100 μs, and 300 μs delays before response collection (new Fig. 3). The data revealed little to no\nemission at the FP emission λ in the 250−400 nm excitation range for either the FPs alone or\nmax\nTb3+ alone for either delay condition. However, when the supercharged FPs were combined with\nTb3+, they exhibited a long-lived emission across the same excitation range. These results provide\ndirect evidence of both (i) sensitizing interactions within the electronically manipulable\n18\nframework of the protein interior and (ii) a near-UV range stimulus resulting in the excitation of\nlanthanide cations and subsequent transfer of energy to the protein biosensor.\nReviewer #1 (Remarks to the Author):\nI have had an opportunity to go through the comments and responses by the authors. I am quite happy\nwith the responses and they adequately address the comments I raise, particularly with respect to figure\ndetails and some of the text clarifications.\nReviewer #2 (Remarks to the Author):\nI would like to thank the authors for addressing some of my concerns from the first round of review.\nHowever, there are still some matters, particularly around selectivity, that remain to be addressed.\nLine 37: “defunct” is not a good word to use here. The lanthanide could be directly excited, albeit\ninefficiently…it is not “dead”\nLine 60: “its superlative nature could limit its utility as a biosensor at greater than picomolar scales of\nlanthanide occupancy in more concentrated environmental streams.” This is a misunderstanding,\nbecause the sensor would likely be used at micromolar concentrations (similar to the concentrations the\nauthors use in their experiments), in which case it would be able to detect lanthanides in the nanomolar\nor micromolar range. Please revise.\nLine 98: As I mentioned in my first review, although the authors have softened their language by\nremoving claims of novelty in favor of “underutilized,” I do feel that the referencing obscures the\nsimilarity of the authors’ approach to previous work such as ref. 36. Rather than relegating it to one of\nthree references for a rather vague sentence at the beginning of the results, the authors should mention\nprevious uses of their strategy in the introduction. It is important to give the reader a real perspective of\nhow the authors’ work relates to prior work.\nLine 148: “florescence” should be “fluorescence”\nFigure 3: I think it would be better, instead of normalizing the maximum signal in each panel to 1, to\nnormalize all to the same value (some intensity from the 0 s delay spectrum). This will make clearer how\nquickly the total signal is decaying over time.\nLine 181 and Figure 4: The authors say that they see response with yttrium. Do they mean ytterbium\n(which they say they tested in line 118)? If they do see a response to yttrium, perhaps the sensing\nmechanism is distinct from that which they claim?\np. 13: talking about seeing no effect of interferents at 1 nM does not make sense. The protein\nconcentration is 3.7 μM in these experiments, so realistically, even if the sensors were very tight binding,\nno response or interference would be seen until roughly the high nanomolar range, and almost half of\nthe experimental points are below this value. While I’m sure it is unintentional, presenting the results in\nthis way is a bit deceptive. It is true that Al3+ gives minimal interference under these conditions, but the\nexperiment with iron showed approximately 50% decrease (SI figure 8, relative to the results in Figure 4b\nsince 0.005 is the baseline value – calling this a 15% or 23% decrease is not really correct). This means\nthat the apparent Kd for iron binding to the sensor is roughly the same as for lanthanide ions. Also, do\nthe authors know that the iron is still in the 2+ state? Given what is written in the methods, I would\nguess that the iron has been oxidized almost immediately to 3+. This may explain the interference.\nSimilarly, in SI figures 6 and 8, as mentioned in my first review, giving metal concentrations as nanomolar\nis confusing to the reader. The fact that half of the experimental points are in the 1 to 1000 nM range\ntakes a moment to figure out with axes in nanomolar.\np. 14 experiments with Virginia Canyon solutions (Fig 5, SI figure 10). Again, not seeing interference in\nexperiments where 1 mM Tb has been added but there is less than a stoichiometric (vs. the number of\nmetal binding sites) amount of potential interferents is not a very stringent test. Are the components of\nthese simulated source materials described anywhere? It looks to me that Cu2+ is a significant\ninterference for the GFP sensor but less of a problem for the YFP sensor. However, the authors should\nnote that Tris is not a good buffer to use because it binds transition metals tightly, especially Cu2+ (which\nis probably the biggest metal ion of concern as an interference because it tends to bind tightly to\nligands). In other words, using Tris decreases the free concentration of potentially interfering metals\naccessible to the sensor which would tend to overestimate claims of selectivity. HEPES or MOPS are\nbetter choices. These buffers may have very weak interactions with lanthanides, but this effect would be\nless important than the impact of Tris being present. Therefore, some of the selectivity experiments\nshould be revisited.\nLine 338: 10 nM is the lower limit of detection of LaMP1, not the upper limit of detection. The lower\nlimit of detection of the FP sensors in the present work is somewhere between 10 and 100 micromolar\nunder ideal conditions. Under more complex conditions, most of the tests were done with\nsubstoichiometric or equimolar amounts of Tb and potential interferents, at best. So I think it may best\njust to say that the present sensors might have a place in detecting high (millimolar) concentrations of\nTb, although tests in more complex solutions will be necessary.\nIn a lot of process streams there are far higher concentrations of non-luminescent lanthanides like La\nthan of Tb, Tm, etc. As I mentioned before, I think it is important to see how these lanthanides interfere\nwith detection of the luminescent lanthanides.\nReviewer #3 (Remarks to the Author):\nThe authors have adequately addressed Reviewers' comments.\nResponse to Reviewer #1:\nReviewer #1 (Remarks to the Author):\nI have had an opportunity to go through the comments and responses by the authors. I am\nquite happy with the responses and they adequately address the comments I raise,\nparticularly with respect to figure details and some of the text clarifications.\nWe thank Reviewer #1 for their time and thoughtful feedback, which allowed us to improve the\nquality of our work.\nResponse to Reviewer #2:\nReviewer #2 (Remarks to the Author):\nI would like to thank the authors for addressing some of my concerns from the first round\nof review. However, there are still some matters, particularly around selectivity, that\nremain to be addressed.\nWe thank Reviewer #2 for their detailed review of our manuscript and for their additional,\ninsightful feedback. Below, we provide a comprehensive point-by-point response for their\nconsideration.\nLine 37: “defunct” is not a good word to use here. The lanthanide could be directly\nexcited, albeit inefficiently…it is not “dead”\nWe thank the Reviewer for calling to attention the inexactness of the use of ‘defunct’ in this\ncontext. The line in question now reads:\nLine 38: In other words, the organic ligand operates as an antenna, transducing energy to an\notherwise sub-operative lanthanide beacon.\npicomolar scales of lanthanide occupancy in more concentrated environmental streams.”\nThis is a misunderstanding, because the sensor would likely be used at micromolar\nconcentrations (similar to the concentrations the authors use in their experiments),\nin which case it would be able to detect lanthanides in the nanomolar or micromolar range.\nPlease revise.\nWe thank the Reviewer for suggesting a more accurate characterization of the LanM-based\nsensor. Indeed, this sensor could likely be used at micromolar concentrations. The claim,\nwhich had previously suggested otherwise, has now been removed, and furthermore, we have included\nan endorsement of LanM’s potential usage in the micromolar range:\nLine 60: Although LanM confers an ultra-high sensitivity to the fluorescence-based detection of\nlanthanides, its superlative nature could limit its utility as a biosensor in more concentrated\nenvironmental streams. Considering that acid mine drainage and other waste streams can\ncontain an upwards of 20–100 µM of total REEs, a sensitivity range congruent with such\nconditions, whether achieved via LanM or some alternative approach, is needed for practical\nbiosensing applications.\nLine 98: As I mentioned in my first review, although the authors have softened their\nlanguage by removing claims of novelty in favor of “underutilized,” I do feel that the\nreferencing obscures the similarity of the authors’ approach to previous work such as\nref. 36. Rather than relegating it to one of three references for a rather vague sentence\nat the beginning of the results, the authors should mention previous uses of their strategy\nin the introduction. It is important to give the reader a real perspective of how the authors’\nwork relates to prior work.\nWe thank the Reviewer for underscoring the importance of expanding upon our discussion of\nprior related work. Indeed, the use of the lanthanide-to-chromophore modality of energy\ntransfer bears similarity to the method described by Rajapakse et al. We would like to point out,\nhowever, that the work by Rajapakse et al. exploits the interaction between trimethoprim and\ndihydrofolate reductase to bring the lanthanide and the chromophore within signaling range. Our\nmethod (i.e., the use of raw surface charge), seen in the context of the binding interaction, is\n2\ndeserving of the claim of novelty. Still, we agree with the Reviewer about the importance of\nhighlighting the similarity in the modality of energy transfer, and we have now made the\nfollowing addition to the Introduction, referencing the work by Rajapakse et al. directly:\nLine 82: Following an underutilized lanthanide-to-chromophore pathway of energy transfer\n(Rajapakse et al.), ultraviolet (UV) excitation resulted in a detectable, dose-dependent signal\ntransmission…\nLine 148: “florescence” should be “fluorescence”\nWe thank the Reviewer for pointing this out. The spelling of ‘fluorescence’ is now correct in Line\n153.\nFigure 3: I think it would be better, instead of normalizing the maximum signal in each\npanel to 1, to normalize all to the same value (some intensity from the 0 s delay spectrum).\nThis will make clearer how quickly the total signal is decaying over time.\nWe thank the Reviewer for their suggestion to reframe the data in Figure 3, and we agree that\napplying the same normalization across time delay conditions could make clearer the decay trend\nover time (new Supplemental Figure 5).\n(B) GFP−10 alone, (C) YFP−31:Tb3+, and (D) YFP−31 alone. In the absence of Tb3+, the fluorescence\nsignal decays to ~0, whereas the excitation of Tb3+ enables a long-lived response, detected via\nfluorescence spectroscopy. From 100 µs to 300 µs, the signal decays further, though the\nTb3+:biosensor energy transfer is still detectable at 300 µs (new Supplementary Fig. 6).\nEqually informative, from a mechanistic perspective, is the direct comparison of signal decay\nbetween the biosensor:Tb3+ condition and the biosensor-only condition at each time interval,\ndisplayed in Figure 3.\nLine 181 and Figure 4: The authors say that they see response with yttrium. Do they mean\nytterbium (which they say they tested in line 118)? If they do see a response to yttrium,\nperhaps the sensing mechanism is distinct from that which they claim?\nWe are grateful to the Reviewer for noting this detail. Indeed, we tested ytterbium, not yttrium,\nand had mistakenly used the chemical symbol for yttrium. Figure 4 and the corresponding text\nhave now been revised:\nLine 188: Additional testing with the lanthanides Eu3+, Sm3+, and Yb3+ revealed…\np. 13: talking about seeing no effect of interferents at 1 nM does not make sense. The\nprotein concentration is 3.7 μM in these experiments, so realistically, even if the sensors\nwere very tight binding, no response or interference would be seen until roughly the high\nnanomolar range, and almost half of the experimental points are below this value.\ninterferents at 1 nM. Indeed, it is somewhat superfluous to underscore the absence of\ninterference at 1 nM in the text, considering that the protein concentration is 3.7 µM, although\nwe believe it is important to display the complete titration from 1 nM to a molar excess of\ninterferent in the Figures. AMDs can be processed to remove iron and aluminum, albeit\nimperfectly (Goodman et al., 2023). Therefore, we believe that our results concerning\neven nanoscale amounts of interferents are highly relevant to the real-world applicability of our\nbiosensors. To minimize superfluity, we have now removed in-text elaborations concerning the\ninterferents at 1 nM.\nIt is true that Al3+ gives minimal interference under these conditions, but the experiment\nwith iron showed approximately 50% decrease (SI figure 8, relative to the results in\nFigure 4b since 0.005 is the baseline value – calling this a 15% or 23% decrease is not\nreally correct).\nWe thank the Reviewer for calling to attention the inaccuracy of our estimates of the % decrease.\nIndeed, the baseline response is not 0. We have now removed our estimates of the % decrease\nand instead characterize the response to 100 µM and equimolar Fe2+ as a ‘modest diminution’\nand ‘disruption’, respectively:\nLine 233: …concentrations greater than 10 µM resulted in a modest diminution of the signal\nresponse. At 1 mM of Fe2+, the signal weakened even more, indicating that an equimolar\nconcentration of Fe2+ in the environment could disrupt the detection of lanthanides.\nAlso, do the authors know that the iron is still in the 2+ state? Given what is written in\nthe methods, I would guess that the iron has been oxidized almost immediately to 3+.\nThis may explain the interference.\nIndeed, the iron could have been oxidized to its 3+ state. We have now added the following\ncaveat:\nreadily oxidized to Fe3+.\nSimilarly, in SI figures 6 and 8, as mentioned in my first review, giving metal\nconcentrations as nanomolar is confusing to the reader. The fact that half of the\nexperimental points are in the 1 to 1000 nM range takes a moment to figure out with axes\nin nanomolar.\nWe thank the Reviewer for their suggestion to improve the clarity of the axes. Supplementary\nFigure 8 and Supplementary Figure 10 (formerly Supplementary Figures 6 and 8) now read in\nµM.\np. 14 experiments with Virginia Canyon solutions (Fig 5, SI figure 10). Again, not seeing\ninterference in experiments where 1 mM Tb has been added but there is less than a\nstoichiometric (vs. the number of metal binding sites) amount of potential interferents is\nnot a very stringent test.\nWe thank the Reviewer for their concern about the relevance of testing interference under\nconditions where Tb3+ is present in stoichiometric excess relative to the interferent. As stated\npreviously, various strategies exist to remove iron and aluminum (Goodman et al., 2023), as well\nas copper, manganese, and other interferents (Elgoud et al., 2022) from lanthanide mixtures.\nTherefore, we believe that data concerning the response, or lack thereof, to a wide range of\nconcentrations of interferents (whether in excess to the lanthanide or not) is especially important\nfor biosensing applications involving real-world outflows, pre- and post-refinement.\nWith respect to the stringency of our test, in addition to assessing responses to nano- and\nmicroscale amounts of interferents, we also tested the response to equimolar and excess\ninterferents relative to Tb3+ (Supplementary Fig. 11).\nAre the components of these simulated source materials described anywhere? It looks to me that\nCu2+ is a significant interference for the GFP sensor but less of a problem for the YFP sensor.\nline, modeled after the compositions of AMDs described by Goodman et al.:\nLine 254: With fidelity to the ratio of contaminants, we subjected GFP−10:Tb3+ and YFP−31:Tb3+\nto mixtures of Al3+, Fe2+, Mn2+, Zn2+, and Cu2+, termed ‘VC’, as well as Mn2+, Zn2+, and Cu2+,\ntermed ‘mVC’.\nZn2+ Cu2+ Mn2+ Al3+ Fe2+ Total Interferents\nVirginia Canyon (mM) 3.79 0.32 2.50 3.33 0.06 10\nModified Virginia Canyon (mM) 11.47 0.97 7.56 0 0 20\nWith respect to Cu2+ interference, indeed, we also noted that Cu2+ is less of an issue for the YFP\nsensor than it is for the GFP sensor (Supplementary Fig. 11). We have included the following line\nto make note of this interesting finding:\nLine 248: Interestingly, Cu2+ appeared to interfere more significantly with Tb3+ detection by the\nGFP sensor compared to the YFP sensor.\nHowever, the authors should note that Tris is not a good buffer to use because it binds\ntransition metals tightly, especially Cu2+ (which is probably the biggest metal ion of\nconcern as an interference because it tends to bind tightly to ligands). In other words,\nusing Tris decreases the free concentration of potentially interfering metals\naccessible to the sensor which would tend to overestimate claims of selectivity. HEPES or MOPS are\nbetter choices. These buffers may have very weak interactions with lanthanides, but this\neffect would be less important than the impact of Tris being present. Therefore, some of\nthe selectivity experiments should be revisited.\nWe thank the Reviewer for their concern about the choice of buffer for the interference assays.\nPrior to conducting the experiments, we noted the predilection of Tris for binding transition\nmetals. Simultaneously, we noted the predilection of HEPES, MOPS, and MES for scavenging\nlanthanides, particularly at buffer concentrations most suitable for our assays (~10−50 mM).\ncomplexed to the buffer (Mandal et al., 2022). Contrast that with Tris, with which no\ncomplexation was observed at 10 mM of buffer. Therefore, despite the ability of Tris to bind\ntransition metals, we saw it fit to prioritize the availability of the lanthanide. The use of HEPES or\nMOPS would likely produce erroneous results, leading to an underestimation of the lanthanide–\nprotein interaction. In general, it is our opinion that HEPES and MOPS should be avoided in the\ninvestigation of lanthanides.\nLine 338: 10 nM is the lower limit of detection of LaMP1, not the upper limit of detection.\nThe lower limit of detection of the FP sensors in the present work is somewhere between\n10 and 100 micromolar under ideal conditions. Under more complex conditions, most of\nthe tests were done with substoichiometric or equimolar amounts of Tb and potential\ninterferents, at best. So I think it may best just to say that the present sensors might have\na place in detecting high (millimolar) concentrations of Tb, although tests in more\ncomplex solutions will be necessary.\nWe thank the Reviewer for pointing this out. 10 nM is indeed the lower limit of detection for 5\nnM of LaMP1. We have now revised the line in question to read as follows:\nLine 346: In contrast, the lanthanide biosensor LaMP1, composed of a FRET donor–acceptor pair\njoined by a collapsible LanM arm, has a considerably higher affinity for lanthanides…\nAdditionally, we have made the following revisions in the Discussion to summarize the potential\nutility (and one particularly salient limitation) of the present sensors, given the lower limit of\ndetection of 10–100 µM:\nLine 343: At present, the platform’s operational scope encompasses the environmentally and\nindustrially significant micromolar to millimolar range…\nmultiple competing metals, will necessitate additional study, and may require more aggressive\nrefinement to be made compatible with the present sensors.\nIn a lot of process streams there are far higher concentrations of non-luminescent\nlanthanides like La than of Tb, Tm, etc. As I mentioned before, I think it is important to\nsee how these lanthanides interfere with detection of the luminescent lanthanides.\nWe thank the Reviewer for your suggestion to test interference with non-luminescent\nlanthanides. Indeed, the non-luminescent lanthanide lanthanum is often present in high\nconcentrations in REE-rich process streams. Lutetium, on the other hand, is typically far less\nabundant than the luminescent lanthanides in various AMD inputs (Goodman et al., 2023). We\nhave now tested La3+ in a competitive assay with 1 mM of Tb3+ and found that a 5-fold excess of\nLa3+ interfered minimally with Tb3+ detection by (A) GFP–10 and (B) YFP–31 (new Supplementary\nFig. 15).\nLine 261: In addition to transition metal contaminants, REE-rich process streams often contain a\nhigh concentration of the non-luminescent lanthanide lanthanum (La3+). Upon measuring the\ndose response of La3+ in a competitive assay with GFP−10:Tb3+ and YFP−31:Tb3+, we observed no\nsignificant perturbation of Tb3+ detection at a 5-fold excess of La3+ relative to Tb3+.\nResponse to Reviewer #3:\nThe authors have adequately addressed Reviewers' comments.\nsignificantly improved our manuscript.\n10"}]

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