Ranmodulin orthologs with improved rare earth separation performance

Abstract

A protein that binds to rare earth metals is provided. The protein may have improved rare earth metal / rare earth metal selectivity. The protein may have four EF-hand motifs, each having 11, 12, or 13 amino acid residues. Each EF-hand motif is separated by 12 or 13 amino acid residues, each amino acid residue is any canonical amino acid residue, and at least one amino acid residue is a hydrophobic amino acid residue. When the EF-hand motif has 12 amino acid residues, the motif may have the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 Also provided are devices and kits comprising the proteins of the present disclosure. Methods of using the proteins and devices are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 376,060, filed September 16, 2022, and U.S. Provisional Application No. 63 / 505,052, filed May 30, 2023, the disclosures of which are incorporated herein by reference.

[0002] [Statement Regarding Federally Sponsored Research] This invention was made with government support under Grant Nos. DE-SC0021007 and DE-AC52-07NA27344 awarded by the Department of Energy, Grant No. CHE-1945015 awarded by the National Science Foundation, and Grant No. GM119707 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] [Sequence table] This application contains a Sequence Listing that has been submitted in .xml format and is incorporated herein by reference in its entirety. The .xml copy was created on September 18, 2023, is named "074339_00251_ST26.xml", and is 114,140 bytes in size. Background of the Disclosure

[0004] The irreplaceable role of rare earth (RE) elements in ubiquitous modern technologies, from permanent magnets to LEDs and phosphors, has sparked renewed interest in one of the grand challenges of separation science: the efficient separation of lanthanides. The separation of these 15 elements is based on the similar physicochemical properties of the predominant +III ions (ionic radii of La). III and Lu IIIThe separation of REs is complicated by the fact that the separation factor between adjacent REs decreases by only 0.19 Å, which also leads to the coexistence of these metals in rare earth-bearing minerals. Traditional hydrometallurgical liquid-liquid extraction methods for RE production utilize organic solvents such as kerosene and toxic phosphonate extractants, and require tens to hundreds of steps to obtain high-purity individual RE oxides. The inefficiency of RE separation and its significant environmental impact have led to research into alternative ligands with larger separation factors between adjacent REs, as well as greener process designs that achieve RE separation in fewer steps and use all-aqueous chemistry.

[0005] The discovery of the founding member of the lanmodulin (LanM) family of lanthanide-binding proteins demonstrated that nature has evolved a macromolecule that surpasses the selectivity of synthetic f-element chelators. The prototypical LanM, a small (12 kDa) monomeric protein from Methylorubrum extorquens AM1 (Mex-LanM), exhibits selective conformational responses toward picomolar concentrations of lanthanides and actinides, facilitating lanthanide uptake in methylotrophs and serving as a technological platform for the detection, recovery, and separation of f-elements. Unusually among rare earth chelators, Mex-LanM selectively binds larger and more abundant light rare earth elements (LREs), particularly La, over heavy rare earth elements (HREs). III -Sm III I prefer. Summary of the Invention

[0006] The present disclosure provides proteins that bind rare earth metals, devices and kits that include the proteins of the present disclosure, and methods of using the proteins and devices.

[0007] In one aspect, the present disclosure provides proteins that bind metals (e.g., lanthanides and / or actinides). Other metal binding proteins are disclosed in WO2020051274 and WO2023004333, which are incorporated herein by reference.

[0008] Proteins of the present disclosure can be of various lengths. For example, proteins of the present disclosure have 65 to 160 amino acid residues (including all integer amino acid values ​​therebetween and ranges therebetween). For example, proteins have a molecular weight of approximately 8 kDa to 14 kDa (including all 0.1 Da values ​​therebetween and ranges therebetween) (e.g., ∼12 kDa). Proteins of the present disclosure include at least one segment capable of binding one or more rare earth metals. In various examples, the segment has at least 70% homology (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology) with the sequence of Hansschlegelia quercus LanM (sometimes referred to as Hans-LanM). In various other examples, the protein is truncated. For example, the protein is truncated at the N-terminus by deletion of the first 10, 20, 30, or 40 residues of the entire translated sequence. In another example, the protein is truncated at the C-terminus by deletion of the last 10, 20, 30, or 40 residues of the entire translated sequence. In various examples of truncated sequences, EF hands 2 and 3 remain, as do most of the hydrophobic core of the protein.

[0009] In various examples, a protein or peptide of the present disclosure capable of dimerizing upon contact with a metal / metal ion may include four EF-hand motifs (e.g., a first EF-hand motif, a second EF-hand motif, a third EF-hand motif, and a fourth EF-hand motif), each of which includes 11, 12, or 13 amino acid residues (e.g., 12 amino acid residues). Each EF-hand motif is separated by 12 or 13 amino acid residues, each of which is any canonical amino acid residue, and at least one amino acid residue is a hydrophobic amino acid residue. In the case of the third EF-hand motif (i.e., EF3) and the fourth EF-hand motif (i.e., EF4), they are separated by the sequence (X)5-R-(X)6, where each X is any canonical amino acid residue. When the EF-hand motif has 12 amino acid residues, the motif can have the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E In the first EF hand motif (i.e., EF1), the second EF hand motif (i.e., EF2), and EF4, X 1 is D or N; X 3 is D, N, or E; X 5 is D, N, or E; X 8 is a hydrophobic residue; X 9 is D, E, or T; X 10 is a hydrophobic residue; and X 2 , X 4 , X 6 , X 7 , and X 11 are each independently any canonical amino acid residue. In various examples, X of EF1, EF2, and / or EF4 7 is T or S. In EF3, X1 is N;X 3 is D;X 4 is G or A;X 5 is D or N; X 7 is T or S;X 8 is a hydrophobic residue; X 9 is E;X 10 is a hydrophobic residue; X 11 is D; and X 2 and X 6 are each independently any canonical amino acid residue (e.g., NX 2 -DX 4 -X 5 -X 6 -X 7 -X 8 -EX 10 -DE (SEQ ID NO: 97). In various examples, X of EF3 4 is A. In various examples, X in EF3 8 is L. In various examples, X of EF3 10 is L, I, or M. Without intending to be bound by any particular theory, it is believed that the dimerization strength of such proteins depends on the identity of the bound metal ion, with dimers preferentially forming in the presence of trivalent rare earth elements or actinides. A protein having this sequence can be linked to another protein of the present disclosure via a peptide linker as described herein.

[0010] In various examples, proteins or peptides of the present disclosure may have enhanced REE / REE selectivity. The selectivity may be between light and heavy rare earth metals and may be higher than the selectivity of M. extorquens lanmodulin. Such proteins may include four EF-hand motifs (e.g., a first EF-hand motif, a second EF-hand motif, a third EF-hand motif, and a fourth EF-hand motif), each EF-hand motif comprising 11, 12, or 13 amino acid residues, each EF-hand motif separated by 12 or 13 amino acid residues, each residue being a canonical residue, and at least one amino acid residue being a hydrophobic amino acid residue. When the EF-hand motif has 12 amino acid residues, the motif may have the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E The first EF-hand motif (i.e., EF1) and the fourth EF-hand motif (i.e., EF4) contain X 1 is D or N; X 3 is D, N, or E; X 5 is D, N, or E; X 8 is a hydrophobic residue; X 9 is D, E, or T; X 10 is a hydrophobic residue; and X 2 , X 4 , X 6 , X 7 , and X 11 are each independently any canonical amino acid residue. In various examples, X of EF1 and / or EF4 7 is T or S. In EF2, X 1 is N;X 3 is D;X 5 is D;X 7is T or S; X 8 is a hydrophobic residue; X 9 is E;X 12 is E; and X 2 , X 4 , X 6 , X 10 and X 11 are each independently any canonical amino acid residue (e.g., NX 2 -DX 4 -DX 6 -X 7 -X 8 -EX 10 -X 11 -E (SEQ ID NO: 95). In various examples, X of EF2 8 is L, I, M, or V. In EF3, X 1 is D;X 3 is D;X 5 is D;X 6 is G;X 7 is T or S; X 8 is a hydrophobic residue; X 9 is D; and X 2 , X 4 , X 10 , and X 11 are each independently any canonical residue (e.g., DX 2 -DX 4 -DGX 7 -X 8 -DX 10 -X 11 -E (SEQ ID NO: 96). In various embodiments, X of EF3 8 is L, I, M, or V. At least one X in either EF2 or EF3 2 is P. A protein having this sequence can be linked to another protein of the disclosure via a peptide linker as described herein.

[0011] In one aspect, the present disclosure provides a device, the device comprising one or more proteins of the present disclosure.

[0012] In one embodiment, the present disclosure provides a kit. The kit may provide one or more proteins of the present disclosure and / or one or more devices of the present disclosure. The kit may include instructions for use of the proteins or devices.

[0013] In one aspect, the present disclosure provides various methods of using the proteins and / or devices of the present disclosure, which may be for binding one or more lanthanides and / or actinides or for detecting and / or quantifying the amount of one or more lanthanides and / or actinides.

[0014] The method of the present disclosure may be a method for detecting and / or quantifying the amount of one or more lanthanides and / or actinides in a sample. The method may include contacting the sample with one or more proteins and / or devices of the present disclosure. The contacted sample may then be exposed to light to obtain a luminescence result of the exposed, contacted sample. The obtained luminescence result may then be compared to a known standard curve for the particular lanthanide or actinide. The concentration may then be determined by the comparison. The known standard curve may be prepared based on the desire to detect and / or determine the amount of any particular lanthanide or actinide. Methods for preparing a standard curve are known in the art.

[0015] A method using the proteins and / or devices of the present disclosure can be a method of binding one or more rare earth metals (e.g., lanthanides and / or actinides) in a sample. Binding can occur by contacting the sample with one or more proteins and / or devices of the present disclosure. The method can be performed on various types of samples. Examples of samples include, but are not limited to, drinking water, wastewater, groundwater, ash ponds, aqueous extracts from contaminated soils, wastewater (e.g., mine drainage such as acid mine drainage), or leachates (e.g., e-waste leachates or ore leachates). In various other examples, the sample is a solid sample. The method can be applied to samples with various pH values. For example, the sample has a pH of 6 or less (e.g., 5.5 or less, 5 or less, 4.5 or less, 4 or less, 3.5 or less, or 3 or less). In various examples, the pH is greater than 6.

[0016] Various lanthanides (e.g., lanthanide ions) and / or actinides (e.g., actinide ions) may be bound by the protein and / or device. For example, the lanthanide is selected from Tb, Eu, Dy, Sm, Nd, and ions thereof. In various examples, the lanthanide is Tb or its ion. The bound lanthanides and / or actinides may be the same or different. The concentration of the lanthanide and / or actinide in the sample may be less than 1 ppm. [Brief explanation of the drawings]

[0017] For a more complete understanding of the nature and objects of the present disclosure, reference is made to the following examples taken in conjunction with the accompanying drawings.

[0018] Figure 1 shows that Hans-LanM diverges from Mex-LanM in sequence and RE / RE selectivity. A: The sequence similarity network (SSN) of the core LanM sequences shows that Hans-LanM forms a distinct cluster. The SSN contains 696 LanM sequences connected by 48,647 edges, with a BLAST E-value of 1 × 10 -5 The threshold for sequence identity is 65%. Black boxes surround nodes clustered with Hans-LanM. The Mex-related LanM sequence (▼) and four sequences within Hansschlegelia (▲) are expanded relative to other nodes (○). The color of the node indicates the family of origin of the sequence. B: Comparison of the four EF-hand sequences of Mex- and Hans-LanMs. C;Hans-LanM's La III Circular dichroism spectra obtained from a representative titration with La, showing metal-related conformational reactions that increase helicity. Apoprotein is in bold black, La III -Saturated proteins are bold red D;Hans-LanM(La III , Nd III , Dy III ) CD titration (pH 5.0). Each point represents the mean ± standard deviation of three independent experiments. E: Comparison of Kd,app values ​​(pH 5.0) between Mex-LanM and Hans-LanM (plotted against ionic radius). Mean ± standard error from three independent experiments.

[0019] FIG. 2 shows the dimerization equilibrium sensitive to the LRE versus HRE or non-RE configuration. a, Apparent molecular weight of the Hans-LanM complex containing the RE, determined by analytical SEC (solid line) or SEC-MALS (dashed line). See Table 1 for conditions. Each data point is the result of a single experiment. b: La determined by X-ray crystallography III -Bound Hans-LanM dimer. La III The ion is spherical, Na I The ions are grey spheres. c) Detailed view of the dimer interface near EF3 in chain A (schematic diagram). Arg100 in chain C (schematic diagram) interacts with Asp93 in chain A and two EF3 La III The hydrogen-bond network involving the ligands (Glu91 and Asp85) anchors these interactions, which constitute the only polar contacts at the dimer interface and provide a means to control the radius of the lanthanide-binding site in EF3. d: Schematic of interactions at the dimer interface. Dashed lines indicate hydrogen-bonding interactions, while other dashed lines indicate hydrophobic contacts. e;La III -bond (left) and Dy III DENSS projection of the electron density from the SAXS data set of the Hans-LanM bond (right) and the dimer La III - PyMOL-generated ribbon diagram of the Hans-LanM crystal structure overlaid with the

[0020] Figure 3 shows that Hans-LanM uses an extended hydrogen-bonding network to control lanthanide selectivity. a;La III -Enlarged view of EF2 and EF3 in Hans-LanM. III Ions are shown as green spheres. Coordination and hydrogen bonds are shown as dashed lines. Residues contributed by chain A are shown, as are residues contributed by chain C (in the case of EF3). (Inset) La III -Hans-LanM and Dy III -Hans-LanM superposition showing the carboxylate shift of Glu91. b;Nd III Representative metal binding site (EF3) in -Mex-LanM. Nd III Ions are shown as spheres. Solvent molecules are shown as spheres.

[0021] Figure 4 shows how Hans-LanM can be utilized to separate Nd / Dy in a single-step process. a) Hans-LanM and the R100K variant showed a greater difference in the stability of the Nd:Dy complex against citrate-induced desorption than Mex-LanM. Mean ± standard error of three independent experiments. III [Citrate] 1 / 2 **Significant differences between La III The effect of dimerization on complex stability is shown (p<0.01, ANOVA [Bonferroni post-hoc test]). Data for Nd and Dy in Mex-LanM are from Dong et al., ACS Cent. Sci. 7, 1798, 2021. b; Spectrofluorimetric titration (λ) of Hans-LanM and R100K variants at pH 5.0 ex =280nm, λ em = 333 nm), showing desorption of a 2:1 metal:protein complex by malonate. Mean ± standard error of three independent experiments, except for R100K, which was a single experiment for each condition. c) Partition coefficients of immobilized Hans-LanM, R100K-Hans-LanM, and Mex-LanM (pH 5.0, ~0.33 mM each RE, La III -Dy III ) Comparison. Each point represents the mean ± standard deviation of three independent experiments. d) Separation of a 95:5 mixture of Nd:Dy using immobilized R100K-Hans-LanM and desorption scheme with three concentrations of malonate followed by HCl at pH 1.5. The bed volume was 0.7 mL.

[0022] Figure 5 shows a sequence alignment of Mex-LanM and Hans-LanM (after signal peptide removal), showing 33% sequence identity. The EF-hands are shown in bold. See Table 12 for the full-length Hans-LanM sequence, including the predicted signal peptide. The Hans-LanM protein used in this study consists of residues A24-K133. The sequence shown is as follows: APTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFKDLDPDKDGTLDAKELKGRVSEADLK (SEQ ID NO: 99), ASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWA (SEQ ID NO: 100), KLDPDNDGTLDKKEYLAAVEAQFKAANPDNDGTIDARELASPAGSALVNLIR (SEQ ID NO: 101), and RANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK (SEQ ID NO: 102)

[0023] Figure 6 shows the La III and Dy III The stoichiometric metal titration of Hans-LanM (15 μM) with RE is shown and monitored by CD spectroscopy. Molar ellipticity at 222 nm is plotted against the equivalent amount of metal added. Experimental conditions: 20 mM acetate, 100 mM KCl, pH 5.0. Each data point is the mean ± SD of two independent determinations. Taken together with Figure 1, these observations support the stoichiometric metal titration of Hans-LanM with RE. III We suggest the following model for the interaction with ions: strong metal binding (with preference for the LRE) at the first site leads to a conformational change; the second site responds to the LRE (cooperatively with the first) to provide a complete conformational response, but responds noncooperatively to Dy only at concentrations >0.5 μM; and the third site does not induce observable conformational changes with any RE. Based on previous studies with Mex-LanM and the crystal structure described herein, the third site is inferred to be EF1; EF2 and EF3 cannot be clearly distinguished as the first and second sites. This more complex response profile than Mex-LanM appears to be tailored to ensure a complete and cooperative response only to the LRE.

[0024] Figure 7 shows the apo Hans-LanM (black) and 3.0 equiv. of La III The size-exclusion chromatogram of metallated Hans-LanM (red) is shown. The S75 column volume was 24 mL. Apoproteins such as apo-Mex LanM elute over a broad molecular weight range (30-70 kDa), suggesting multiple promiscuous conformations. III The bound protein exhibited a soluble, high molecular weight species formed when high concentrations of RE were added to Hans-LanM, and also showed a symmetrical peak at approximately 28 kDa, suggesting a dimer.

[0025] Figure 8 shows the RE III Size-exclusion chromatography of Apo-Hans-LanM complexes (RE = La, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Y) is shown. Apo-Hans (590 μM, 100 μL) was metallated with 3.0 equivalents of RE (mixed 0.5 equivalents at a time) and loaded onto a 24 mL analytical S75 column calibrated as described. a; The chromatograms for RE=La to Dy show apparent molecular weights suggestive of Hans-LanM dimers for La and Nd, but this gradually shifts to lower apparent molecular weights, which is thought to indicate a decrease in the proportion of dimers in rapid equilibrium with the monomer. b; Beyond Dy, the speciation of the complex is complex but suggests the emergence of a more extended monomer or dimer population with slower exchange rates.

[0026] Figure 9 shows the SEC-MALS traces of La-, Nd-, and Dy-conjugated Hans-LanM, showing the slower elution and lower weight-average molar mass of the Dy conjugate (see Table 3). Apo-Hans-LanM was added to 3 equivalents of each RE. III After incubation with ions, precipitates and aggregates were removed by centrifugation and analytical SEC, and the protein was injected onto the column at concentrations of 114–128 μM.

[0027] FIG. 10 shows the dimer dissociation of apoHans-LanM (ITC). (Top) Representative ITC trace when 300 μM protein was titrated into buffer. (Bottom) Thermograms obtained from the above data were fitted to a dimer dissociation model using NanoAnalyze software (parameters shown in Table 18). Conditions: 30 mM MOPS, 100 mM KCl, pH 7.0, 30°C.

[0028] Figure 11 shows the Dy III 2-Hans-LanM dimer dissociation (ITC). (Top) Representative ITC trace when 300 μM protein was titrated into buffer. (Bottom) Thermograms obtained from the above data were fitted to a dimer dissociation model using NanoAnalyze software (parameters shown in Table 18). Conditions: 30 mM MOPS, 100 mM KCl, pH 7.0, 30°C. Using Equation 1, the dimer dissociation rate of Dy under SEC-MALS conditions was III - The concentrations of Hans-LanM monomer and dimer can be calculated ([P] = 18.9 μM (obtained from Table 17), K dimer = 60 μM (obtained from ITC) (Table 18). [M] = 13.5 μM, and therefore [D] = 3.3 μM. Because [D] is approximately 25% of [M], this result corresponds well with the average mass of this form obtained by SEC-MALS of 15.5 kDa, which is approximately 25% higher than the expected MW (11.9 kDa), suggesting partial dimerization. This supports our interpretation that the SEC-MALS results indicate a rapid monomer-dimer equilibrium in which the individual monomer and dimer components cannot be separated and therefore appear as a weighted average of the two populations.

[0029] Figure 12 shows the La III 2-Hans LanM titration (ITC). (a, top) Representative ITC trace upon titration of 150 μM protein into buffer. (a, bottom) Thermogram obtained from the above data. (b, top) Representative ITC trace of 540 μM protein titrated into buffer. An initial 0.2 μL injection was followed by nine 5.0 μL additions. (b, bottom) Thermogram obtained from the above data. In these experiments, the difference in heat between each injection cannot be observed, so La III This highlights the extremely tight binding of the bound dimer. Conditions: 30 mM MOPS, 100 mM KCl, pH 7.0, 30°C. Using Equation 1, the peak concentration from SEC-MALS, [P] = 18.4 μM (Table 17), and conservatively assuming a minimum threshold of observable monomer [M] of 10% (1.84 μM) of the total protein concentration, La III The maximum dimer dissociation constant of apo-Hans-LanM can be estimated because it appears in SEC-MALS as a difference of approximately 1 kDa from the theoretical monomer molecular weight of 11.9 kDa (such a difference is observable in the case of apo-Hans-LanM, see Table 17). In this case, K dimer Solving this gives 0.4μM, which is La III It represents the maximum dissociation constant of the dimer of the binding protein. III In the presence of Dy III This shows a 100-fold or greater enhancement of dimerization compared to the presence of ATP.

[0030] FIG. 13 shows a comparison of the topology and key residues in Hans- and Mex-LanMs. a, La III -Hans-LanM and Nd III -Topology diagram of Mex-LanM. Despite only 33% sequence identity, the overall topology of the two proteins is very similar, with three core helices (α1-3) forming a central three-helix bundle, decorated with two auxiliary helices preceding EF1 and EF3. As illustrated in Figure 32, EF4 of Mex-LanM has Nd III The presence of ions is a result of the high protein concentration used for crystallization. b, Sequence alignment of the key regions of Hans- and Mex-LanM with H. sapiens calmodulin. Similarities in the metal-binding residues in these proteins are highlighted in light blue. Note the presence of a Glu residue at position 9 in each EF-hand of Hans-LanM, which is unique compared to other proteins. Residues involved in interactions at the dimer interface of Hans-LanM are shown in bold (hydrogen-bonding interactions: E91, D93, R100) (hydrophobic interactions: I43, I47, H48, T63, M92, L96). The charges of the residues corresponding to D93 and R100 in Hans-LanM are reversed in Mex-LanM (K94, E101). Several residues involved in hydrophobic interactions in Hans-LanM (I43, I47, L96) correspond to similar residues in Mex-LanM (L44, L48, L97), whereas other residues (H48, T63, M92) correspond to residues without bulky side chains (A49, G64) or to charged residues (K94). This analysis also highlights another potentially important difference: calmodulin has highly conserved Gly residues at both positions 4 and 6 of each EF-hand. Gly residues are conserved at these positions in 81–100% of predicted calmodulin sequences, depending on the EF-hand / residue location. In contrast, Hans- and Mex-LanM EF-hands have glycines at only one of these positions (none at all in Hans-LanM EF1). The sequences shown are NKDNDDSLEIAEVIH (SEQ ID NO:114), NPDGDTTLESGE (SEQ ID NO:103), DPDKDGTIDLKEALA (SEQ ID NO:104), DPDKDGTLDAKE (SEQ ID NO:105), DKDGDGTITTKEIII (SEQ ID NO:106), DADGNGTIDFPE (SEQ ID NO:107), NKDGDQTLEMDEWLKILR (SEQ ID NO:108), DANKDGKLTAAE (SEQ ID NO:109), DPDNDGTLDKKEYLAAVE (SEQ ID NO:110), NPDNDGTIDARE (SEQ ID NO:111), DKDGNGFISAAE (SEQ ID NO:112), and DIDGDGQINYES (SEQ ID NO:113).

[0031] Figure 14 shows the raw SAXS data of La-, Nd-, and Dy-bound Hans-LanM. SAXS data sets were measured in 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0, in the presence of different metal ions on an in-house Rigaku BioSAXS2000. nano The SEC-MALS fractions were collected as above. a. The La-binding protein was 1.5 mg / mL, the Nd-binding protein was 1.4 mg / mL, and the Dy-binding protein was 1.6 mg / mL. SAXS data sets were collected over 60 minutes using an autosampler quartz flow cell with six 10-minute images. SAXS data for buffer were collected for 60 minutes using the same flow cell and used for reference subtraction. An overlay of the six 10-minute images averaged in each case showed no damage from X-ray radiation. b. and c. CRYSOL (ATSAS) fits of the SAXS data of La and Dy to the crystallographic La-Hans-LanM and Dy-Hans-LanM dimer models. Chi-squared values ​​are shown in Table 6.

[0032] Figure 15 shows an overlay of Guinier plots for La-, Nd-, and Dy-bound Hans-LanM. The fits reveal radii of gyration (R) of 18.5 ± 2.5 Å (La), 18.7 ± 2.7 Å (Nd), and 17.8 ± 2.6 Å (Dy). g ) (see Table 4). The R of Dy and La / Nd complexes is higher than expected based on SEC-MALS. g Note that there is no difference between the values ​​(Table 17 suggests a difference of about 2-3 Å in the hydrodynamic radii of La / Nd and Dy). This is because the protein concentration used for SAXS is five times higher than for SEC-MALS, resulting in a significantly higher population of Dy-bound dimers in the SAXS experiment. Nevertheless, these differences in RE dependence are due to the SAXS R g The values ​​are within the uncertainty range.

[0033] Figure 16 shows the La IIIThe combined Hans-LanM solvent envelope is shown. Density from Solution Scattering (DENSS) is an algorithm that calculates ab initio electron density maps from solution scattering data. The DENSS electron density map, which appears as a transparent surface in the PyMOL-generated display, shows the dimer La, color-coded by chain (light blue and green). III -Hans-LanM crystal structure. III Ions are represented as gold spheres. The color gradient goes from least electron dense to most electron dense: blue (2σ) → cyan (5σ) → green (7.5σ) → yellow (10σ) → red (15σ). Manual fitting of the envelope and model was performed in PyMOL. The CRYSOL fit of the calculated SAXS profile is overlaid with the experimental SAXS profile (chi-squared fit 1.1) (Table 6). The left panel shows the La shown in Figure 2e. III -Same as Hans-LanM data.

[0034] Figure 17 shows the Nd III The DENSS electron density map, which appears as a transparent surface in the PyMOL-generated display, shows the dimer La III -Hans-LanM crystal structure (colored by chain (light blue and green)), La III The calculated SAXS profile is overlaid with the experimental SAXS profile (Chi-squared fit 1.3) (Table 6). Manual fitting of the envelope and model was performed in PyMOL.

[0035] Figure 18 shows the Dy III The DENSS electron density map is shown as a transparent surface in the PyMOL-generated display, and the dimer La III -There is little overlap with the crystal structure of Hans-LanM. The CRYSOL fit of the calculated SAXS profile of the dimer is superimposed with the experimental SAXS profile (chi-squared fit 3.8) (Table 6).

[0036] Figure 19 shows the distance distribution (P(r)) analysis, suggesting that Hans-LanM transitions from a single species with La and Nd to two states with Dy. Overlay of the pairwise distance distribution function P(r) for the La (green), Nd (blue), and Dy-coupled (red) Hans-LanM data set. The R obtained from this analysis g The values ​​-18.5 Å (La), 18.7 Å (Nd), and 17.6 Å (Dy) - are the R values ​​obtained from the Guinier analysis. g For the La and Nd complexes, P(r) has a bell shape, indicative of scattering from spherical particles. For Dy, P(r) shows three shoulders, suggesting mixed species (e.g., a mixture of monomers and dimers).

[0037] Figure 20 shows Kratky plots suggesting increasing flexibility of Hans-LanM in the order La → Nd → Dy. Kratky plots of Hans-LanM with La bond (green), Nd bond (blue), and Dy bond (red). Kratky plots obtained from SAXS data provide qualitative information about the flexible and / or folded state of a protein. The gradual departure from the q-axis from La to Nd to Dy bonded states suggests increasing disorder, which is due to the weak Dy bond. III This may be related to reduced binding and cooperativity (Figure 1d) and / or to the monomer-dimer equilibrium. q is expressed in Å -1 .

[0038] Figure 21 shows the La III Joint Hans-LanM EF-Hand 3 and 4 2F o -F c Electron density map (gray mesh outlined at 1.0σ) and anomaly difference map (purple mesh outlined at 3.0σ) are shown. a, In EF4, the solvent is clearly visible, and the lack of anomalous differential density is due to La III does not occupy this site. Metal ions are not fully occupied by Na for the reasons explained in Figure 52. Iwas modeled as. b, For EF3, which represents EF hands 1-3, the electron density map shows no solvent coordination, and the anomalous difference density map shows fully occupied La III coincides with the ion.

[0039] FIG. 22 shows the structure of the LanM EF hand in comparison with the EF hands of calmodulin (CaM) and lanthanide-dependent methanol dehydrogenase (MDH). a, La III -Binding Hans-LanM EF2. La III The ions are green spheres. b, Nd III EF3 of Mex-LanM binding. III Ions are light blue spheres, and coordinating solvent molecules (w1, w2) are red spheres. c, EF2 of H. sapiens CaM (PDB code: 1CLL). Ca II Ions are grey spheres, and coordinating solvent molecules are red spheres. d, La III The active site of the lanthanide-dependent MDH, XoxF, from Methylomicrobium buryatense 5GB1C metallated with 10-coordinate La (1.85 Å resolution, PDB code: 6DAM). III Ions are shown as green spheres. The enzyme also requires a pyrroloquinoline quinone cofactor (PQQ). Nd III -Mex-LanM and Ca IIThe primary coordination spheres of Mex-LanM and Mex-LanM are nearly identical, except that the D5 residue is monodentate in CaM (Asp24) but bidentate in Mex-LanM (Asp88), and Mex-LanM has an extra water molecule (w1). These images also highlight the consequence of Mex-LanM having an Asn at position 1 of the EF-hand (N1), whereas Mex-LanM and CaM have an Asp (D1). This substitution results in different peptide backbone structures between positions 4 and 6 of the EF-hand. In Hans-LanM, the non-coordinating side chain Nδ of Asn58 hydrogen bonds with the backbone CO of the Asp62 residue (position 5), whereas in Mex-LanM and CaM, the non-coordinating atom is oxygen, allowing a hydrogen bond with the backbone NH of the residue at position 6. This difference is explained by the fact that Hans-LanM has a Gly at position 4 of EF hands 2 and 3, whereas Mex-LanM has a Gly at position 6 and CaM has a Gly residue at both positions (Fig. 13b).

[0040] Figure 23 shows the Eu III Spectroscopic estimation of the coordinated solvent molecule (q) of 2-Hans-LanM. III The luminescence lifetime of the complex is empirically correlated with q. ▲: Value of the luminescence decay time constant (τ, unit ms, left Y axis). For comparison, τ H2O = 1.24ms is the value for Hans-LanM, but only 0.404ms for Mex-LanM. ●: 1 / τ value (unit: ms -1 , right y-axis). A fit equation of 1 / τ and mole fraction DO is used to determine q. The uncertainty in q is assumed to be ±0.5. A value of q of 0.11 is consistent with the absence of coordinating solvent in the Hans-LanM crystal structure. Conditions: 20 μM Hans-LanM, 40 μM Eu III , 25 mM HEPES, 75 mM NaCl, pH 7.0. Each data point is the mean ± sd of duplicate independent samples.

[0041] Figure 24 shows the extended hydrogen bond network in the metal binding sites of Hans-LanM and Mex-LanM. a, The metal site of Hans-LanM displays extensive hydrogen bonds between the ligand and several backbone amides, as well as the side chain of the Thr residue at position 7. In EF3, this network is further extended by interactions between Asp85 and Glu91 with Arg100 of the neighboring monomer. b, The metal site of Mex-LanM features a similar hydrogen-bonding pattern, but solvent molecules w1 and w2 replace Glu69. c, Close-up of the backbone flip that allows hydrogen-bonding interactions between the Asp and Asn residues at position 1, the main-chain amide and the carbonyl in Mex-LanM and Hans-LanM, respectively.

[0042] Figure 25 shows the La III , Nd III , Dy III The titration of R100K-Hans-LanM with Nd is shown (pH 5.0) using CD spectroscopy. The fit parameters are summarized in Table 7. III and Dy III Results using (K d,app and the change in molar ellipticity) were essentially the same as those obtained with the wild-type Hans-LanM protein; III When using K d,app The kinetics of the ATPase was reduced by two-fold, and the change in molar ellipticity was increased. Conditions: 15 μM protein, 20 mM acetate, 100 mM KCl, pH 5.0, 10 mM EDTA (La, Nd) or EGTA (Dy), 0-10 mM RE. III Each data point is the mean ± sd of two independent samples.

[0043] Figure 26 shows the results of the synthesis of 3 equivalents of La as apoprotein. III Figure 7 shows the SEC-MALS trace of R100K-Hans-LanM metallated with La. The apoprotein migrates similarly to Mex-LanM and wild-type Hans-LanM (Figure 7), suggesting that the protein is disordered. IIIThe complex migrates as a single sharp peak. Both samples had similar weight-average molar masses determined by MALS corresponding to the monomer (see Table 8). Conditions: 3 mg / mL protein, 30 mM MOPS, 100 mM KCl, pH 7.0.

[0044] Figure 27 shows that Hansschlegelia quercus LanM and LanM proteins, predicted to dimerize, are phylogenetically distinct from other LanMs. A Bayesian phylogeny was constructed using a site-homogeneity model based on a Whelan and Goldman matrix with invariant sites and four distinct gamma categories (WAG+I+Γ4) under a strict clock with a minimum sequence length of 106 amino acids. Monophyletic groups containing members of the Hans cluster are highlighted in gray. Node values ​​indicate posterior probabilities after 10,000,000 iterations with a 25% burn-in. The scale bar represents a change of 0.1 per amino acid position. The LanM core sequence alignment used to construct the phylogeny is colored using the Zappos scheme. The four EF-hand domains are indicated by lines at the bottom of the alignment, and residues involved in dimer interactions are marked with asterisks. A Bayesian phylogeny constructed from this alignment supports the network structure, where the Hans cluster is represented as a monophyletic group and is positioned distantly from other sequences. Furthermore, the topology of the Hans cluster in the phylogenetic tree corresponds to the proximity seen in the network (Fig. 1a and Fig. 5). In this alignment, R100 is at position 87, indicated by an asterisk, along with three other residues in EF-hand 3 involved in the dimerization interface (i.e., D72, E78, and D80). All four residues involved in dimerization are conserved in the Hans cluster LanM, suggesting that these proteins all form dimers. Only a single LanM outside the Hans cluster (unclassified Hyphomicrobiaceae) has an Arg residue at position 87. The EF3 sequence of this ortholog is similar to several Ln IIIAlthough lacking a ligand, EF2 mediates the interaction with Arg in Hans (in EF3). 11 The residues featured in this alignment suggest that this unidentified LanM may form a dimer along a distinct interface. None of the sequences in this alignment contain a basic amino acid at position 87; instead, most contain an acidic residue (usually Glu), as in Mex-LanM. Furthermore, a group of LanMs containing two cysteine ​​residues (near each end) (proximal to EF1 and EF4) was identified. Given that LanM is a periplasmic protein, it is conceivable that these proteins may have disulfide bonds between these residues, which may enhance the structural stability of LanM, although further evidence is needed to confirm the role of these residues.

[0045] Figure 28 shows the Dy III -Two views of Hans-LanM's EF hand 3, showing the 2Fo-Fc electron density map (gray mesh outlined at 1.0σ) and the anomaly difference map (purple mesh outlined at 3.0σ). a, Density associated with the metal ligand is clear and no coordinating solvent is observed. b, The hydrogen bond network between EF3 and Arg100 (of the adjacent monomer) is also clearly visualized.

[0046] FIG. 29 shows the X-ray absorption edge of Dy-Hans-LanM detected by fluorescence excitation.

[0047] Figure 30 shows the Dy III -Hans-LanM crystal of Dy L III The anomalous diffraction data set collected at the EF edge (7793.5 eV) is shown, supporting the identity of the bound lanthanide as Dy, an HRE. The anomalous difference electron density map is shown with a purple mesh (contoured at 4.0 σ) for a representative metal binding site in chain A. In all four EF hands, L IIIAbove the edge, a fairly strong anomalous difference electron density map peak was observed (Table 10). Interestingly, EF2 and EF3 showed the largest anomalous difference map peaks, likely reflecting that only two high-affinity sites were biochemically observed in this complex (Fig. 1d, Fig. 6).

[0048] Figure 31 shows the Dy III All four copies of the Arg100-EF3 hydrogen bond network in the asymmetric unit of -Hans-LanM showed the same shift of Glu91 to a monodentate configuration, and the hydrogen bond between this residue and Arg100 was III -Hans-LanM from about 2.9 Å to Dy III The Arg100-Asp93 hydrogen bond is also shown to be longer than the La-Hans-LanM bond, at 3.2 Å. III The Arg100-Asp85 hydrogen bond is longer than that of the La-Hans-LanM, from 2.5 Å to 2.7 Å. III -Hans-LanM 3.2Å to Dy III -Hans-LanM was slightly compressed to 2.8 / 2.9 Å. Although forced dimerization under high-concentration conditions for crystallography may alter the interactions between monomers compared to low-concentration solutions, the La- and Dy-bound structures show how the carboxylate shift of Glu91 alters this second-sphere hydrogen-bonding network, possibly preventing dimerization. This explanation is strongly supported by the characterization of the R100K variant (Table 8, Figures 25-27).

[0049] Figure 32 shows a Nd III 1 shows the X-ray crystal structure of bound Mex-LanM. a) Overall structure of Mex-LanM. Nd in EF4 III The ions were prepared under the crystallization conditions (3.5 equiv. Nd III and millimolar proteins); prior biochemical analysis has shown that the weakest binding corresponds to a micromolar K d , and the weak site was shown to be associated with EF4. b, 2Fo-Fc electron density map (gray mesh outlined at 1.0σ) and anomalous difference map (purple mesh outlined at 3.0σ) of EF3. Two solvent molecules are Nd III It can be seen that the Nd ions are coordinated. III Ions are light blue spheres and solvent molecules are red spheres. c, Details of the four EF hands. The metal coordination in EF1-3 is the same, the backbone CO of D1, D3, and T7 is a monodentate ligand, and D5 and E 12 is a bidentate ligand, and two water molecules (w1, w2) are nine-coordinated. In EF4, the D3 residue (Asp110) is bidentate. Because EF4 has an Asn instead of an Asp at position 1, the uncoordinated side chain N cannot hydrogen bond with the backbone, which is thought to lower the affinity of this site.

[0050] Figure 33 shows La III -Hans-LanM and Nd III A comparison of the hydrogen bond networks connecting the metal binding site and the exit helix in -Mex-LanM is shown. Extending the backbone CO-HN hydrogen bond network within each helix to include the metal site is a key step in RE III This may contribute to the overall stabilization of the folded state of the protein in the -LanM complex. a, La III In HRE-Hans-LanM, the E9 residue is a bidentate ligand, so a direct analogous hydrogen bond does not occur, but Glu91 of EF3 is connected to the first backbone NH of the exit helix through a hydrogen-bonding network involving Arg100 and Asp93. Disruption of this network in the presence of HRE, and therefore disruption of the bond between the helix and the metal site, may also contribute to the low stability of the HRE-Hans-LanM complex. b, Nd III In -Mex-LanM, the D9 residue (Asp92 in EF3) connects the Glu95 NH of the exit helix to the Nd IIIThe difference in the length of these two hydrogen bonds resulting from the coordination of different REs may contribute to the selectivity trend of Mex-LanM.

[0051] Figure 34 shows La III Representative fluorescence emission intensities and wavelengths (λ) obtained from single measurements of wild-type Hans-LanM during titration at 1000 kJ / s. max ) changes. a, Hans emission spectrum (λ ex =278nm). La III Binding results in a two-fold increase in intensity, λ (max) shifts from 343 nm to 333 nm. b, Excitation and emission wavelengths suggest that two Trp residues in the protein near EF2 and EF3, Trp79 and Trp95, contribute primarily to the spectrum. Trp95 corresponds to Tyr96 in Mex-LanM (Figure 5), and its fluorescence intensity has been shown to be sensitive to metal binding and / or associated conformational changes in the protein. Conditions: 20 μM protein, 30 mM MOPS, 100 mM KCl, pH 7.0.

[0052] Figure 35 shows typical Nd breakthrough curves for Hans-LanM and R100K-Hans-LanM immobilized on a column. In the experiment, 0.4 mM Nd was immobilized in 7 mM homo-PIPES, pH 5.0. III The Hans-LanM column immobilized 4.4 ± 0.08 μmol / mL of protein, with a Nd adsorption capacity of 4.6 ± 0.23 μmol / mL (1.06 equivalents). The R100K-Hans-LanM column immobilized 3.2 ± 0.06 μmol / mL of protein, with a Nd adsorption capacity of 6.66 ± 0.33 μmol / mL (2.08 equivalents). Each protein was immobilized once; the uncertainty in the immobilized protein concentration represents the standard deviation of triplicate protein concentration measurements by BCA assay. The uncertainty in the adsorption capacity was assumed to be 5%, based on our previously reported experiments with the LanM column.

[0053] FIG. 36 shows that the Hans complex with the HRE precipitates more readily than the LRE complex. (A) Photograph of Hans+Dy (left) and La (right); (B) Photograph of the sample in (A) after centrifugation; (C) Photograph of the sample in (A) after redissolution with EDTA. This property may be applicable to separation procedures.

[0054] Figure 37 shows the apparent K of 15 μM Hans-LanM. d Values ​​are shown relative to His-tagged Mex-LanM (monitored using CD spectroscopy). Buffer: 30 mM acetate, 100 mM KCl, 0-10 mM Ln III , 10 mM EDTA or EGTA, pH 5.0.

[0055] FIG. 38 (left) CD spectrum of Mex-LanM I42L / N108D / I115L (10 μM) showing that the protein exhibits additional α-helical content in the apo state compared to the wild-type protein. (Right) CD spectrum of Mex-LanM I42L / N108D / I115L (5 μM) was measured using CD spectroscopy in various EDTA-buffered free Nd III Ion concentrations were monitored. Buffer: 20 mM acetate, 100 mM KCl, 0-10 mM Nd III , 10 mM EDTA, pH 5.0. Although there is some imprecision in the ellipticity due to the low concentration in a single accumulation, the data are in the 1 pM range of K d,app This suggests that: (Bottom) Determination of the binding stoichiometry of wild-type Mex-LanM and its variants. III was titrated into a solution of protein and xylenol orange as previously described (Cotruvo et al., JACS 2018). The absorbance at 574 nm was measured as Nd III Plotted against equivalent weight.

[0056] Figure 39 shows the results of various EDTA-buffered free NdO2 samples using CD spectroscopy. IIICD titration curve (left) and [θ] of 20 μM Mex-LanM(A32D / A117K) monitored by ion concentration. 222nm (Right) Buffer: 30 mM acetate, 100 mM KCl, 0-10 mM Nd III , 10 mM EDTA, pH 5.0. Apparent K d = 20 ± 1 pM, n = 1.85 ± 0.08. d,app Because the points at and below are at the low end of the EDTA buffering range (<2% "% high solution" value, or 200 μM total Nd), metal binding to the 20 μM protein may have influenced the free metal concentration but was not taken into account in the calculation. Therefore, the true K in this experiment d,app The values ​​are slightly overestimated (i.e., the affinity is underestimated).

[0057] Figure 40 shows the results of various EDTA-buffered free NdO2 samples using CD spectroscopy. III CD titration curve (left) and [θ] of 20 μM Mex-LanM(A32D / A117R) monitored by ion concentration. 222nm (Right) Buffer: 30 mM acetate, 100 mM KCl, 0-10 mM Nd III , 10 mM EDTA, pH 5.0. Apparent K d = 22 ± 1 pM, n = 1.51 ± 0.08. d,app Because the points at and below are at the low end of the EDTA buffer range ("High Solution %" values ​​of <2%, or 200 μM total Nd), metal binding to the 20 μM protein may have influenced the free metal concentration but was not taken into account in the calculations. Therefore, the true K in this experiment was not considered. d,app The values ​​are slightly overestimated (i.e., the affinity is underestimated).

[0058] Figure 41 shows Eu IIITime-resolved (left) and steady-state (right) fluorescence emission spectra comparing LanM_001 (Mex-LanM) and LanM_002 (Hans-LanM) bound to [protein] = 20 μM, [metal] = 60 μM. Buffer: 30 mM MOPS, 100 mM KCl, pH 7.0. λ ex =280nm, delay=150μs (left). λ ex =280nm(right).

[0059] Figure 42 shows the Tb III Time-resolved (left) and steady-state (right) fluorescence emission spectra comparing LanM_001 and LanM_002 bound to [protein] = 20 μM, [metal] = 60 μM. Buffer: 30 mM MOPS, 100 mM KCl, pH 7.0. λ ex =300nm, delay=100μs (left). λ ex =280nm(right).

[0060] Figure 43 shows the Sm III (Left) and Dy III (Right) Time-resolved fluorescence emission spectra comparing LanM_001 and LanM_002 bound to the protein. [Protein] = 20 μM, [Metal] = 60 μM. Buffer: 30 mM MOPS, 100 mM KCl, pH 7.0. λ ex =280nm, delay=150μs (left). λ ex =290nm, delay=125μs (right).

[0061] Figure 44 (left) 4.0 equivalents of La III or Dy III Size-exclusion chromatography of 20 μM LanM_012 in the presence of ATP. A sharp peak at a retention volume of approximately 12.0 mL indicates a dimeric species with an apparent molecular weight of approximately 29.1 kDa under both conditions. Buffer: 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0. (Right) Size-exclusion chromatography of 400 μM LanM_012 in the absence of lanthanide. The peak at a retention volume of approximately 14.0 mL suggests a monomeric species with an apparent molecular weight of approximately 14.2 kDa. Buffer: 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0.

[0062] Figure 45 shows the results of competition with the indicator xylenol orange, in which LanM_012 reacts with 2.0 equivalents of La III The results show that the protein binds tightly to the ATP (monitored at 575 nm). Protein: 20 μM, Buffer: 20 mM MES, 100 mM KCl, 5 mM acetate, pH 6.0.

[0063] Figure 46 shows the effect of 20 μM LanM_012 on the ATP synthesis at pH 5.0. III The titration of 0 Eq was followed using circular dichroism spectroscopy. The spectrum of the apoprotein (0 Eq) suggests a well-folded protein with significant α-helical content. Upon addition of metal, the shape of the curve shifted slightly around 205 nm, suggesting ordering of the loop regions and only minor changes in the overall secondary structure of the protein. These preliminary results indicate that the apo form of the protein is already well-folded. Buffer: 20 mM acetate, 100 mM KCl, pH 5.0.

[0064] Figure 47 shows CD spectra of LanM_012 in the apo form or complexed with 2.0 equivalents of La or Dy. The temperature was increased at 2°C / min, and spectra were collected after each increase. Spectra at 16°C, 28°C, 74°C, and 84°C were plotted for Apo (A), La (B), and Dy (C). The signal between 218 nm and 222 nm was averaged for each spectrum obtained and plotted against temperature (D). The dotted line indicates the ellipticity value of the apoprotein at 222 nm at high temperature. Protein: 20 μM, Buffer: 30 mM MOPS, 100 mM KCl, pH 7.0.

[0065] Figure 48 (left) Tryptophan fluorescence titration of 10 μM LanM_012 followed in the presence of various free metal concentrations. Buffer: 20 mM acetate, 100 mM KCl, 0-10 mM Ln III pH 5.0. Free La III and Dy III The concentrations were buffered with 10 mM EDTA and 10 mM EGTA, respectively. ex =280nm. (Right) LanM_012 (20 μM) was metallated with 3.0 equivalents of La, Nd, or Dy, and tryptophan fluorescence at 333 nm was monitored in the presence of various citrate concentrations. Dissociation of the protein-metal complex caused a loss of tryptophan fluorescence intensity. Buffer: 20 mM acetate, 100 mM KCl, pH 5.0. λ ex =280nm.

[0066] Figure 49 shows the Sm III (A) and Dy III (B) Time-resolved fluorescence emission spectra comparing apo LanM_012 and LanM_012-Ln2. III (C) and Tb III (D) Steady-state fluorescence emission spectra comparing apo LanM_012 and LanM_012-Ln2. [Protein]=20 μM, Buffer: 30 mM MOPS, 100 mM KCl, pH 7.0. λ ex = 280 nm, delay = 150 μs (A); λ ex = 290 nm, delay = 125 μs (B); λ ex = 280 nm (C); λ ex =280nm(D).

[0067] Figure 50 is an expanded view of the inset of Figure 1a (the Hans cluster), which contains 20 sequences and 190 edges. The Hans cluster includes LanMs from bacteria of the genera Hansschlegelia, Ancylobacter, Methylopila, Oharaeibacter, Starkeya, and Xanthobacter. While these genera are restricted to this cluster, family-level members are found dispersed throughout the network, including 1 Xanthobacteraceae and 42 Methylocystacea.

[0068] Figure 51 shows (a) Ca II , (b) Nd III , (c) Dy III The Hans-Lan CD titration is shown in Figure 1 using a chelating agent buffer solution of Dy. III (maximum 0.3 μM) and Ca II (Maximum 5.5mM) III and La III induces a similar, incomplete conformational change in the protein compared to the conformational change induced by The data in the right panel of a are representative titrations from the three data sets used to generate the plot in the left panel. Data in b and c are representative titrations from the three data sets used to generate the plot in Figure 1d. Conditions: 15 μM protein, 20 mM acetate, 100 mM KCl, 10 mM EDTA (for Ca and Nd titrations) or EGTA (for Dy titrations), 0-10 mM metal ions. Each data point in (a, left panel) is the mean ± sd of three independent determinations.

[0069] Figure 52 shows the La resolved at 1.8 Å resolution. III The X-ray crystal structure of bound Hans-LanM is shown. a, Overall structure of the asymmetric unit consisting of two Hans-LanM dimers and two citrate molecules in the crystallization solution. The structure of each monomer in the dimer is a Y structure in which EF hands 2 and 3 are paired and EF hands 1 and 4 are paired. III This is consistent with the NMR solution structure of bound Mex-LanM. be, La III Details of metal coordination in the four EF-hands of the Hans-LanM. La in EF-hands 1, 2, and 3 III The coordination sphere of the ion is the side chain Oδ (monodentate) of N1, D3, D5, E9, E 12 The coordination number is 10, consisting of a carboxylate side chain (all bidentate) of La and a backbone carbonyl of S7 (EF1) or T7 (EF2 and EF3). III The distance between the ligands is 2.5 to 2.7 Å. III The crystal radius of is 1.41 Å by Shannon, and the hexacoordinate O 2- Since the radius of La is 1.26Å, III The -O distance is estimated to be 2.57 Å, which is consistent with our results. The metal ion in EF4 is Na, due to the short metal-ligand distance, low coordination number, and the presence of sodium in the crystallization solution. I It was modeled as Ca II Although it cannot be completely excluded because it was present early in the protein purification, when the protein was processed with Chelex at the end of the purification, the crystallographic data were consistent with the Na determined by the CheckMyMetal server. I This ion corresponds to the monodentate D1, N3, and D5 side chains, and the bidentate E 12 It is coordinated in a distorted pentagonal bipyramidal shape by the side chain, the backbone carbonyl of K113, and one solvent molecule, for a total coordination number of 7. I The distances between the lanthanide-ligand and the protein are 2.3-2.5 Å, and 2.7 Å for the solvent molecules. In the case of Mex-LanM, biochemical data and NMR spectroscopy also support the idea that EF4 is a poor lanthanide binding site, and the NMR solution structure is a model without a metal ion.

[0070] Figure 53 shows the Dy III The X-ray crystal structure of bound Hans-LanM is shown. a, One of the dimers of the asymmetric unit consisting of chains A and B. EF4 unexpectedly exhibits Dy III Note that EF1 is occupied only by chain A, whereas EF2 is occupied only by chain B. b, Overall structure of the asymmetric unit consisting of two Hans-LanM dimers. III Unlike the Hans-LanM structure, Dy III In the case of -Hans-LanM, the two dimers and the monomers within each dimer show significant differences. EF2-4 has Dy in all chains. III In chains B and C, no metal ions are bound to the EF-hands, whereas in chain D, Dy is the only metal ion bound to the EF-hands. III Although the ion is bound, the first five residues of EF1 (N34-D38) could not be modeled. III Our decision to model Dy as all four EF-hands is supported by the anomalous diffraction data set (Tables 9-10, Figures 29-30). Biochemical data suggest that in solution, at least one Dy III The binding site is suggested to be weak (see Figure 1d and Figure 6), and studies of Mex-LanM suggest that EF2 / 3 is likely the stronger binding site. This suggestion is supported by the anomalous data for Dy (Table 10), and the occupancy of the weak metal binding site is likely due to the high protein concentration used for crystallography. c, Dy III Details of metal coordination in the EF-hands of -Hans-LanM. In the top row, three different EF1 structures in the asymmetric unit are shown. Only in chain A, the metal site of EF1 is almost identical to that of EF2 and EF3 (La, where the EF1-3 sites are very similar). III -Hans-LanM (Figure 52). In EF1 (chain A), EF2, and EF3, the ligand is La III-Hans-LanM, except that the E9 residues (Glu42, Glu66, and Glu91) are shifted to monodentate and are now nine-coordinate. III The decrease in the coordination number of Dy is consistent with lanthanide contraction and is also observed for other ligands. III The distance between the ligands is mostly 2.3 to 2.5 Å, and La III Consistent with this observation, the 9-coordinate Dy III The crystal radius of La is 1.22 Å, as determined by Shannon. III The carboxylate shift of the Glu residue at position 9 is noteworthy because this position is important for gating affinity and selectivity in other EF-hand proteins. III is seven-coordinated, and La III -Hans sodium site, but with slightly shorter metal-ligand distances (2.2-2.5 Å); again, these distances are closer to those of the heptacoordinate Dy III This is consistent with the predicted value.

[0071] Figure 54 shows spectrofluorimetric titrations of RE-LanM (Hans-LanM, R100K-Hans-LanM, and Mex-LanM) complexes using citrate as a competitor (monitored by intrinsic protein fluorescence). Emission values ​​were normalized to apoprotein fluorescence of 1.0. Note that the fluorescence intensity of the Trp residue in Hans-LanM decreases going from the RE-bound state to the apo state (Figure 34), while the fluorescence intensity of the Tyr residue in Mex increases going from the RE-bound state to the apo state. Initial conditions for all experiments (titration with increasing concentrations of citrate): 20 μM protein, 40 μM RE, 20 mM acetate, 100 mM KCl, pH 5.0. The citrate concentration at which 50% of each metal is desorbed under these conditions ([citrate]) was determined. 1 / 2 ) are summarized in Table 11 and plotted in Figure 4a. a, Hans-LanM. b, R100K-Hans-LanM [Citrate] for La and Nd in wild type vs. R100K-Hans-LanM 1 / 2 The compressed difference between the values ​​is due to the LRE, especially the La III We explain the role of dimerization in enhancing the affinity differential for β-glucan. c, Mex-LanM. Data for Nd and Dy have been reported previously. d, [citrate] relative to Nd for each protein 1 / 2 Value and Dy vs. [citrate] 1 / 2 Comparing the ratios of the values, it can be seen that Hans-LanM has a higher Nd / Dy selectivity than Mex-LanM. The ratios between wild-type Hans-LanM and the R100K variant were not significantly different by two-tailed t-test (p>0.05), suggesting that the hydrogen bond network involving Arg100 is La III Although it has a large effect on selectivity, Nd III This suggests that it does not contribute significantly to / HRE selectivity (Figure 4a). All data are shown as mean ± sd (ac) or standard error (d) of data from three independent experiments.

[0072] Figure 5 shows the separation of a 95:5 mixture of Nd:Dy using immobilized Hans-LanM. The desorption scheme consisted of three malonate concentrations (30, 50, and 90 mM; see right axis) followed by pH 1.5 (HCl). The results revealed that Hans-LanM produced slightly less pure Dy than the R100K variant (83.6% vs. 98% Dy purity, respectively, with comparable yields; compare Figure 4d). In equilibrium binding experiments with La-Dy, a similar selectivity profile was observed for the immobilized proteins from La to Gd, but the selectivity pattern deviated for Tb (Figure 4c). The difference in selectivity between Hans-LanM and the R100K variant was confirmed using the Nd / Dy binary system. Uncertainty in the determination of the partition coefficient for Dy in the nine-element RE group hindered our ability to distinguish small differences in Dy / Nd separation factors between proteins (Tables 13-14). In this Nd / Dy binary experiment (Table 19), the separation factor for Hans-LanM was 8.12 ± 0.40, while that for the R100K variant was 12.7 ± 1.3, consistent with the improved Dy separation effect of R100K. This result is consistent with the values ​​obtained from the nine-element experiment but differs slightly from the equilibrium binding results using free Hans-LanM protein and R100K-Hans-LanM protein, which revealed similarly high selectivity for Nd over Dy (Figure 4a, b). This likely reflects weaker LRE-induced dimerization in the R100K variant at the low protein concentration (20 μM) in the solution experiment using free protein. The La / Nd selectivity on the column also differs from that observed for the apparent Kd values ​​of the free protein (wild-type and R100K) in solution, although the experiments with the free protein used single-element solutions, and mixed-metal binding effects may have influenced the on-column data. The R100K variant also behaves better on the column, as evidenced by the 2:1 RE:protein stoichiometry.One explanation for these results is that immobilization prevents dimerization; however, Figure 2b shows the N- and C-termini of the Hans-LanM dimer, showing that the C-terminus is approximately 20 Å away from the closest part of the dimer interface, suggesting that immobilization itself is not expected to disrupt this interface. However, it must be considered that a functional dimer requires the two C-termini to be immobilized in close proximity, which is unlikely at the immobilization density of our column. Therefore, overall, it appears that the dimerization equilibrium applies only to a small number of protein units immobilized on the column. We speculate that more fully exploiting the dimerization equilibrium in a column format would result in even more robust separations. The most reliable way to obtain a homogeneous dimer population on a column would be to link the two monomers (e.g., by a polypeptide chain), tune the dimerization affinity by mutagenesis of residues that contribute to inter-monomer interactions, and then immobilize this dimer through a single attachment point. Dimerization may also be exploited in other separation formats. These directions are issues we are currently working on.

[0073] Figure 56 shows the SDS-PAGE obtained from S75 gel filtration chromatography of LanM_013, demonstrating high purity. Lanes are labeled with fraction numbers.

[0074] Figure 57 shows that LanM_013 exhibits approximately 2.0 equivalents of La(III) binding by UV-vis spectroscopy after titration. Data at 278 nm are the top points, and data at 285 nm are the bottom points. Buffer: 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0, 20 μM LanM_013.

[0075] Figure 58 shows the LanM_013-metal interaction followed by tracking the tyrosine fluorescence of the protein. Conditions: 20 mM acetate, 100 mM KCl, 5% glycerol, pH 5.0, 20 μM protein.

[0076] Figure 59 shows the results of using citrate as a competitor to remove bound La(III) from LanM_013 (3 equivalents of La(III) added before the start of the experiment). Conditions: 20 mM acetate, 100 mM KCl, 5% glycerol, pH 5.0, 20 μM protein.

[0077] Figure 60 shows the direct assessment of metal binding of LanM_013 at pH 6.0 using xylenol orange competitive titration. Conditions: 20 mM MES, 20 mM acetate, 100 mM KCl, chelex treatment, pH 6.0, 20 μM protein.

[0078] FIG. 61 shows the conformational changes of LanM_013 monitored using circular dichroism spectroscopy. Conditions: (pH 5.0) 20 mM acetate, 100 mM KCl, 5% glycerol, pH 5.0, 20 μM protein. (pH 7.0) 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0, 20 μM protein.

[0079] Figure 62 shows the affinity determination of LanM_013 at pH 5.0. Conditions: 10 μM LanM_013, 20 mM acetate, 100 mM KCl, 0-10 mM LaCl3, 10 mM EDTA, pH 5.0 (left). 20 μM LanM_013, 20 mM acetate, 100 mM KCl, 0-10 mM DyCl3, 10 mM EGTA, pH 5.0 (right). Apparent K d The values ​​are 11 pM (La) and 96 pM (Dy), with Hill coefficients of approximately 1.5 for both.

[0080] Figure 63 shows the steady-state emission spectra of various LanM-based sensors (5 μM) in the presence of two equivalents of Nd(III) (AC) or Yb(III) (DF). (A) Mex-LanM(T90W) with Nd; (B) Hans-LanM (R100K) with Nd; (C) LanM_012 with Nd, (D) Mex-LanM(T90W) with Yb; (E) Hans-LanM (R100K) using Yb (F) LanM_012 with Yb. Excitation 280 nm.

[0081] Figure 64 shows the apparent K of LanM_013 (10 μM) by CD spectroscopy using free lanthanide ion concentrations buffered with EGTA. d s is measured. (A) [θ] 222nm vs Free Gd III Plot of apparent dissociation constant (K d1,app , K d2,app ) and Hill coefficients (n1, n2) were determined using a biphasic fit. (B) [θ] 222nm vs Free Dy III A plot of the apparent dissociation constant (K) was obtained using a single-phase fit. d,app ) and Hill coefficient (n) were calculated. (C) [θ] 222nm vs Free Ho III A plot of the apparent dissociation constant (K) was obtained using a single-phase fit. d,app ) and Hill coefficient (n) were calculated. Buffer: 20mM acetate, 100mM KCl, 0-10mM Ln III -EGTA, pH 5.0. Samples were equilibrated for 3 hours before measurement.

[0082] Figure 65 shows the selectivity profiles of column-immobilized lanmodulin variants for the La-Dy REEs. LanM_001 = Mex-LanM. LanM_002 = Hans-LanM. Comparison of equilibrium distribution (log D) values ​​for all REEs using a LanM column at pH 5. The right plot is the same as the left plot, except that LanM_002 is also included for reference. The calculated separation factors are shown in the table below. Column dimensions: 5 cm x 0.5 cm.

[0083] Figure 66 shows the selectivity profiles of column-immobilized lanmodulin variants for the Gd-Lu,Y REEs. LanM_001 = Mex-LanM. LanM_002 = Hans-LanM. Comparison of equilibrium distribution (log D) of all REEs using a LanM column at pH 5. The right plot is the same as the left plot, except that LanM_002 is also included for reference. The calculated separation factors are shown in the table below. Column dimensions: 5 cm x 0.5 cm.

[0084] Figure 67 shows the characteristics of variant group 1 (A) XO competition assay (pH 6.1) by titration of Nd(III). The absorbance of XO at 574 nm was plotted against the Nd(III) equivalent. (B) Conformational changes of LanM variants (20 μM) were monitored by CD at pH 5.0. Ellipticity at 222 nm was plotted against Nd(III) equivalents.

[0085] Figure 68 shows the characteristics of variant group 2. (A) XO competition assay (pH 6.1) by titration of Nd(III). The absorbance of XO at 574 nm was plotted against the Nd(III) equivalent. (B) Conformational changes of LanM variants (20 μM) were monitored by CD at pH 5.0. Ellipticity at 222 nm was plotted against Nd(III) equivalents.

[0086] Figure 69 shows the characteristics of variant group 3. (A) XO competition assay (pH 6.1) by titration of Nd(III). The absorbance of XO at 574 nm was plotted against the Nd(III) equivalent. (B) Conformational changes of LanM variants (20 μM) were monitored by CD at pH 5.0. Ellipticity at 222 nm was plotted against Nd(III) equivalents.

[0087] Figure 70 shows the characteristics of the "combo" variant group. (A) XO competition assay (pH 6.1) by titration of Nd(III). The absorbance of XO at 574 nm was plotted against the Nd(III) equivalent. (B) Conformational changes of LanM variants (20 μM) were monitored by CD at pH 5.0. Ellipticity at 222 nm was plotted against Nd(III) equivalents.

[0088] Figure 71 shows the conformational change of LanM variant group 3 (5 μM) monitored by CD at pH 5.0. The ellipticity at 222 nm was plotted against the free Nd(III) concentration. (A) A98G (B) A99G (C) A102G Buffer: 20 mM acetate, 100 mM KCl, 10 mM Nd-EGTA, pH 5.0.

[0089] Although the claimed subject matter is described in terms of particular embodiments, other embodiments, including embodiments that do not provide all of the advantages and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes can be made without departing from the scope of this disclosure.

[0090] As used herein, unless otherwise indicated, "about," "substantially," or "such as," when used in connection with a measurable variable (e.g., parameter, amount, duration, etc.) or list of alternatives, is meant to encompass variation of the specified value and variation from the specified value, including, but not limited to, within experimental error (e.g., as may be determined by a particular data set, industry standard, etc., and / or as determined, for example, using a particular confidence interval (e.g., a 90%, 95%, or higher-than-average confidence interval), such as a variation of ±10% or less, ±5% or less, ±1% or less, or ±0.1% or less from the specified value) (to the extent that variation of such variables and / or alternatives is appropriate for practice in this disclosure). As used herein, the term "about" can mean that the amount or value in question is the exact value set forth in the claims or taught herein or a value that provides an equivalent result or effect. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximated and / or larger or smaller, as desired, to reflect tolerances, conversion factors, rounding, measurement errors, etc., or other factors known to those skilled in the art that would yield equivalent results or effects. Generally, a quantity, size, composition, parameter, or other quantity or characteristic, or alternative, is "about" or "e.g., etc.", whether or not expressly stated as such. When "about" is used before a quantitative value, it is understood that the parameter also includes the particular quantitative value itself, unless specifically stated otherwise.

[0091] This specification discloses a range of values. The range has a lower limit and an upper limit. Unless otherwise specified, the range includes the lower limit, the upper limit, and all values ​​between the lower limit and the upper limit, including all values ​​up to the order of magnitude of the minimum value (either the lower limit or the upper limit) of the range, but is not limited thereto. It is understood that such range formats are used for convenience and brevity and should therefore be interpreted flexibly to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges encompassed within the range (as if each numerical value and subrange were explicitly stated). For example, a numerical range of "0.1% to 5%" should be interpreted to include not only the explicitly stated numerical value of 0.1% to 5%, but also individual numerical values ​​(e.g., 1%, 2%, 3%, 4%) and subranges (e.g., 0.5% to 1.1%, 0.5% to 2.4%, 0.5% to 3.2%, 0.5% to 4.4%, and other possible subranges) within the stated range, unless otherwise specified. It is also understood that there are a number of values ​​disclosed herein, and that each value is herein disclosed (as provided above) as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. Similarly, when values ​​are expressed as approximations, the use of the antecedent "about" will understand that the particular value forms a further disclosure. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0092] As used herein, unless otherwise specified, the term "group" refers to a chemical entity that is monovalent (i.e., has one end that can be covalently bonded to another chemical species), divalent, or polyvalent (i.e., has two or more ends that can be covalently bonded to other chemical species). The term "group" also includes radicals (e.g., monovalent and polyvalent, e.g., divalent, trivalent, etc., radicals). Examples of groups include the following: [ka]

[0093] Amino acids and amino acid residues may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0094] Examples of hydrophobic amino acids and hydrophobic amino acid residues include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, proline, cysteine, phenylalanine, methionine, tryptophan, and the like.

[0095] The present disclosure provides proteins that bind to rare earth metals. Also provided are devices and kits that include the proteins of the present disclosure. Also provided are methods of using the proteins and devices.

[0096] In one aspect, the present disclosure provides proteins that bind to metals (e.g., lanthanides and / or actinides). Other metal binding proteins are disclosed in WO2020051274 and WO2023004333, which are incorporated herein by reference.

[0097] The Wt Hans-LanM of the present disclosure may be any of the following peptides: TIFF2025532059000003.tif92169

[0098] Proteins of the present disclosure can vary in length. For example, proteins of the present disclosure have 65 to 160 amino acid residues (including all integer amino acid values ​​and ranges therebetween). For example, proteins have a molecular weight of approximately 8 kDa to 14 kDa (including all 0.1 Da values ​​and ranges therebetween, e.g., approximately 12 kDa). Proteins of the present disclosure include at least one segment capable of binding one or more rare earth metals. In various examples, the segment has at least 70% homology (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology) with the sequence of Hansschlegelia quercus LanM (sometimes referred to as Hans-LanM). In various other examples, the protein is truncated. For example, the protein is truncated at the N-terminus by deletion of the first 10, 20, 30, or 40 residues of the entire translated sequence. In another example, the protein is truncated at the C-terminus by deletion of the last 10, 20, 30, or 40 residues of the entire translated sequence. In various examples of truncated sequences, EF hands 2 and 3 remain, as do most of the hydrophobic core of the protein.

[0099] Suitable Hans-LanM and LanM proteins of the present disclosure include wild-type H. quercus LanM proteins or orthologs from other organisms having at least two EF-hand motifs, at least one of which has at least three carboxylate residues, and at least two of which are separated by a space of 10 to 15 residues. References herein generally to "lanmodulin," "LanM," or "LanM protein" should be understood to include wild-type and orthologs described herein. "LanM" can refer to a complete protein having one or more LanM units, or a portion thereof containing one or more LanM units. A LanM unit contains at least two EF-hand motifs, at least one of which has at least three carboxylate residues, and at least two of which are separated by a space of 10 to 15 residues. For ease of reference, discussions will be made with reference to lanmodulin, LanM, or LanM proteins, and should be understood to include both complete proteins and portions of complete proteins containing the appropriate LanM units.

[0100] Various substitutions may be made in the peptides or proteins of the present disclosure. For example, one or more amino acid residues may be substituted with a different amino acid residue. The amino acid residues may be canonical or non-canonical. For example, R100 may be substituted with an amino acid residue. For example, the arginine may be substituted with a lysine (e.g., R100K). Other suitable variants of Hans-LanM include, but are not limited to, M92L, M92A, M92D, A44N, A44S, A44T, D93N, and D93A. Also included are proteins having at least 70% homology (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology) with any of the aforementioned variants. Residue numbers correspond to the residue numbering when the 23-residue signal peptide is present. For proteins lacking a signal peptide, the first residue of these proteins is residue 24 (after cleavage of the N-terminal methionine).

[0101] In various embodiments, the proteins of the present disclosure have a residue suitable for immobilization to a substrate. The residue can be part of a larger sequence containing 2 to 10 amino acid residues. For example, the residue contains a functional group that chemically reacts with another functional group on the substrate so that the residue (and therefore the protein) is covalently attached to the substrate. For example, the substrate can contain a maleimide group that can react with a nucleophilic group such as the thiol of cysteine ​​or the amine of lysine. Other suitable chemistries (e.g., click chemistry) are known in the art and may be used. For example, the substrate can be a resin or bead containing a functional group that can react with a residue of the LanM protein. For example, the functional group can be a maleimide, alkyne, or azide.

[0102] In various examples, a protein or peptide of the present disclosure capable of binding to a metal / metal ion (e.g., a protein or peptide of the present disclosure capable of dimerizing upon contact with a metal / metal ion) can include four EF-hand motifs (e.g., a first EF-hand motif, a second EF-hand motif, a third EF-hand motif, and a fourth EF-hand motif), each of which includes 11, 12, or 13 amino acid residues (e.g., 12 amino acid residues). Each EF-hand motif is separated by 12 or 13 amino acid residues, each amino acid residue being any canonical amino acid residue, and at least one amino acid residue being a hydrophobic amino acid residue. In the case of the third EF-hand motif (i.e., EF3) and the fourth EF-hand motif (i.e., EF4), they are separated by the sequence (X)5-R-(X)6, where each X is any canonical amino acid residue. When the EF-hand motif has 12 amino acid residues, the motif may have the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E For the first EF hand motif (i.e., EF1), the second EF hand motif (i.e., EF2), and EF4, X 1 is D or N; X 3 is D, N, or E; X 5 is D, N, or E; X 8 is a hydrophobic residue; X 9 is D, E, or T; X 10 is a hydrophobic residue; and X 2 , X 4 , X 6 , X 7 , and X 11 are each independently any canonical amino acid residue. In various examples, X of EF1, EF2, and / or EF47 is T or S. For EF3, X 1 is N;X 3 is D;X 4 is G or A;X 5 is D or N; X 7 is T or S;X 8 is a hydrophobic residue; X 9 is E;X 10 is a hydrophobic residue; X 11 is D; and X 2 and X 6 are each independently any canonical amino acid residue (e.g., NX 2 -DX 4 -X 5 -X 6 -X 7 -X 8 -EX 10 -DE (SEQ ID NO: 97). In various examples, X of EF3 4 is A. In various examples, X in EF3 8 is L. In various examples, X of EF3 10 is L, I, or M. Without intending to be bound by theory, it is believed that the dimerization strength of such proteins depends on the identity of the bound metal ion, with dimers preferentially forming in the presence of trivalent rare earth elements or actinides. A protein having this sequence can be linked to another protein of the disclosure via a peptide linker as described herein.

[0103] In various examples, the proteins or peptides of the present disclosure may have enhanced REE / REE selectivity. The selectivity may be between light and heavy rare earth metals and may be higher than the selectivity of M. extorquens lanmodulin. Such proteins may include four EF-hand motifs (e.g., a first EF-hand motif, a second EF-hand motif, a third EF-hand motif, and a fourth EF-hand motif), each EF-hand motif comprising 11, 12, or 13 amino acid residues, each EF-hand motif separated by 12 or 13 amino acid residues, each residue being a canonical residue, and at least one amino acid residue being a hydrophobic amino acid residue. When the EF-hand motif has 12 amino acid residues, the motif may have the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E The first EF-hand motif (i.e., EF1) and the fourth EF-hand motif (i.e., EF4) contain X 1 is D or N; X 3 is D, N, or E; X 5 is D, N, or E; X 8 is a hydrophobic residue; X 9 is D, E, or T; X 10 is a hydrophobic residue; and X 2 , X 4 , X 6 , X 7 , and X 11 are each independently any canonical amino acid residue. In various examples, X of EF1 and / or EF4 7 is T or S. For EF2, X 1 is N;X 3 is D;X 5 is D;X 7is T or S; X 8 is a hydrophobic residue; X 9 is E;X 12 is E; and X 2 , X 4 , X 6 , X 10 and X 11 are each independently any canonical amino acid residue (e.g., NX 2 -DX 4 -DX 6 -X 7 -X 8 -EX 10 -X 11 -E (SEQ ID NO: 95). In various examples, X of EF2 8 is L, I, M, or V. For EF3, X 1 is D;X 3 is D;X 5 is D;X 6 is G;X 7 is T or S; X 8 is a hydrophobic residue; X 9 is D; and X 2 , X 4 , X 10 , and X 11 are each independently any canonical residue (e.g., DX 2 -DX 4 -DGX 7 -X 8 -DX 10 -X 11 -E (SEQ ID NO: 96). In various embodiments, X of EF3 8 is L, I, M, or V. At least one X in either EF2 or EF3 2 is P. A protein having this sequence can be linked to another protein of the disclosure via a peptide linker as described herein.

[0104] In various embodiments, the second EF hand has the following sequence: X 1 -X 2 -X 3 -X4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E, where X 1 is D or N; X 2 is any canonical amino acid; X 3 is D, N, or E; X 4 is any canonical amino acid; X 5 is D, N, or E; X 6 is any canonical amino acid; X 7 is any canonical amino acid; X 8 is a hydrophobic residue; X 9 is independently D, E, or T; X 10 is a hydrophobic residue; and X 11 is any canonical amino acid. In various other embodiments, the second EF hand has the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E, where X 1 is N; X 2 is any canonical amino acid; X 3 is D; X 4 is any canonical amino acid; X5 is D; X 6 is any canonical amino acid; X 7 is T or S; X 8 is a hydrophobic residue; X 9 is E; X 10 is any canonical amino acid; and X 11 is any canonical amino acid. In various embodiments, the X of the second EF hand 2 is P. In various embodiments, X 7 is T or S. In various embodiments, X 8 is L, I, M, or V.

[0105] In various embodiments, the third EF hand has the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E, where X 1 is N; X 2 is any canonical amino acid; X 3 is D; X 4 is G or A; X 5 is D or N; X 6 is any canonical amino acid; X 7 is T or S; X 8 is a hydrophobic residue; X 9 is E; X 10 is a hydrophobic residue; and X 11 is D. In various other embodiments, the third EF hand has the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E, where X 1 is D; X 2 is any canonical amino acid; X 3 is D; X 4 is D; X 5 is D; X 6 is G; X 7 is T or S; X 8 is a hydrophobic residue; X 9 is D; X 10 is any canonical amino acid; and X 11 is any canonical amino acid. In various embodiments, X 2 is P. In various embodiments, X 4 is A. In various embodiments, X 8 is L, I, M, or V. In various embodiments, X 10 is L, I, or M.

[0106] The proteins of the present disclosure may contain various EF1 hands. Examples of EF1 hands include, but are not limited to, NKDNDDSLEIAE (SEQ ID NO:51), NKDKDSTVEIVE (SEQ ID NO:52), DPDKDGTIDLNE (SEQ ID NO:53), DPDMDNALTLEE (SEQ ID NO:54), DPDKDGTIDLKE (SEQ ID NO:55), DPDKDGTLDLKE (SEQ ID NO:56), NPDHDGTIDWRE (SEQ ID NO:57), DPDGDGAMTLGE (SEQ ID NO:58), NKDNDDSLEAAE (SEQ ID NO:59), NKDNDDSLEVAE (SEQ ID NO:60), NKDNDDSLEINE (SEQ ID NO:61), NKDNDDSLEISE (SEQ ID NO:62), NKDNDDSLEITE (SEQ ID NO:63), and NKDNDDSLQIAE (SEQ ID NO:64).

[0107] The proteins of the present disclosure may contain various EF2 hands. Examples of EF2 hands include, but are not limited to, NPDGDTTLESGE (SEQ ID NO: 65), NPDKDKTLEAAE (SEQ ID NO: 66), NPDGDGTLEVKE (SEQ ID NO: 67), NTDDDNTLEADE (SEQ ID NO: 68), DPDKDGTLDAKE (SEQ ID NO: 69), DPDHDGTLDMKE (SEQ ID NO: 70), NKDGDITLELDE (SEQ ID NO: 71), and NPDGDTTLQSGE (SEQ ID NO: 72).

[0108] The proteins of the present disclosure may comprise various EF3 hands. Examples of EF3 hands include, but are not limited to: NKDGDQTLEMDE (SEQ ID NO:73), NKDGDKTLELDE (SEQ ID NO:74), DPDNDGTLDMQE (SEQ ID NO:75), DPDDDGSLDMAE (SEQ ID NO:76), DPDNDGTLDKKE (SEQ ID NO:77), NPDRDGKLDKHE (SEQ ID NO:78), DLIKGRGISLGE (SEQ ID NO:79), NKDGDQTLELDE (SEQ ID NO:80), NKDGDQTLEADE (SEQ ID NO:81), NKDGDQTLEDDE (SEQ ID NO:82), NKDGDQTLEMAE (SEQ ID NO:83), NKDGDQTLEMNE (SEQ ID NO:84), and NKDGDQTLQMDE (SEQ ID NO:85).

[0109] The proteins of the present disclosure may comprise various EF4 hands. Examples of EF4 hands include, but are not limited to, DANKDGKLTAAE (SEQ ID NO: 86), DANKDGKLTEAE (SEQ ID NO: 87), NPDNDGTVDEKE (SEQ ID NO: 88), NPDGDDTIESDE (SEQ ID NO: 89), NPDNDGTIDKRE (SEQ ID NO: 90), DPDNDGTLDARE (SEQ ID NO: 91), NPDKDGTIDCRE (SEQ ID NO: 92), NPDKDHTIECDE (SEQ ID NO: 93), DPDNDGTIDARE (SEQ ID NO: 94), and NPDNDGTIDARE (SEQ ID NO: 98).

[0110] Examples of proteins of the present disclosure include, but are not limited to: TIFF2025532059000004.tif221169TIFF2025532059000005.tif212169TIFF20255320590 00006.tif234169TIFF2025532059000007.tif237169TIFF2025532059000008.tif223169

[0111] Without intending to be bound by any particular theory, it is believed that at least some of the LanM proteins of the present disclosure dimerize with other LanM monomers upon contact with a rare earth metal. The LanM monomers of a dimer may be the same or different. At least the following sequences are believed to dimerize: TIFF2025532059000009.tif58169 Without intending to be bound by any particular theory, it is believed that the aforementioned sequences may dimerize with or without a signal peptide incorporated into the LanM sequence.

[0112] In various examples, two proteins of the present disclosure can be covalently conjugated. For example, the two proteins can be conjugated via a peptide linker. For example, the linker can be (GGS) n The linker may be a motif, where n is 2, 3, 4, 5, or 6. For example, the linker may be GGSGGSGGSGGSGGSGGS (SEQ ID NO: 43). Examples of conjugate proteins include, but are not limited to, the following: TIFF2025532059000010.tif175169 The foregoing sequences are not intended to be limiting. In various examples, the conjugated proteins of the present disclosure can be immobilized on a resin for use in the devices of the present disclosure.

[0113] Without intending to be bound by any particular theory, it is believed that LanM_013, methyloligella halotolerans, and LanM_011 do not form dimers.

[0114] In one aspect, the present disclosure provides a device, the device comprising one or more proteins of the present disclosure.

[0115] A variety of devices may comprise the proteins of the present disclosure, including, but not limited to, filters, membranes, sensors, portable detectors, plate readers, fluorometers, biosensors, in-line monitors, and the like.

[0116] In one embodiment, the present disclosure provides a kit. The kit may provide one or more proteins of the present disclosure and / or one or more devices of the present disclosure. The kit may include instructions for use of the proteins or devices.

[0117] In one aspect, the present disclosure provides various methods of using the proteins and / or devices of the present disclosure, which may be for binding one or more lanthanides and / or actinides or for detecting and / or quantifying the amount of one or more lanthanides and / or actinides.

[0118] A method using the proteins and / or devices of the present disclosure can be a method of binding one or more rare earth metals (e.g., lanthanides and / or actinides) in a sample. Binding can occur by contacting the sample with one or more proteins and / or devices of the present disclosure. The method can be performed on a variety of sample types. Examples of samples include, but are not limited to, drinking water, wastewater, groundwater, ash ponds, aqueous extracts from contaminated soils, wastewater (e.g., mine drainage, such as acid mine drainage), or leachate (e.g., e-waste leachate or ore leachate). In various other examples, the sample is a solid sample. The method can be applied to samples with various pH values. For example, the sample has a pH of 6 or less (e.g., 5.5 or less, 5 or less, 4.5 or less, 4 or less, 3.5 or less, or 3 or less). In various examples, the pH is greater than 6.

[0119] Various lanthanides (e.g., lanthanide ions) and / or actinides (e.g., actinide ions) may be bound by the protein and / or device. Examples of lanthanides and actinides that may be bound include, but are not limited to, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y, and ions thereof. In various examples, any lanthanide is detected. For example, the lanthanide is selected from Tb, Gd, Ho, Eu, Dy, Sm, Nd, Yb, and ions thereof. In various examples, the lanthanide is Tb, Eu, Sm, Dy, or ions thereof. The bound lanthanides and / or actinides may be the same or different. The concentration of the lanthanide and / or actinide in the sample may be less than 100 ppm (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 1, 0.1, or 0.05 ppm).

[0120] In various examples, one or more lanthanides and / or actinides bound to one or more proteins and / or devices can be isolated and recovered from the proteins and / or devices. The lanthanides and / or actinides can be debound by lowering the pH to below about 2.5 or by adding a chelating agent (e.g., citrate, EDTA, EGTA, malonate, etc.). In various embodiments, when one or more different lanthanides and / or actinides are bound to one or more proteins or devices, the one or more different lanthanides and / or actinides can be sequentially released from the protein. As an illustrative example, when both Nd and Dy are bound, one metal species can be selectively released while the other metal remains bound. For example, one metal can be released by contact with a chelating agent, and the other metal can be released by adjusting the pH. The one or more proteins and / or devices can be reused after the one or more lanthanides have been debound and separated.

[0121] The disclosed method may be a method for detecting and / or quantifying the amount of one or more lanthanides and / or actinides in a sample. The method may include contacting the sample with one or more proteins and / or devices of the disclosed method. The contacted sample is then exposed to light, causing the exposed, contacted sample to luminesce. The resulting luminescence can then be compared to a known standard curve for the particular lanthanide or actinide. The concentration can then be determined by the comparison. The known standard curve can be prepared based on the desire to detect and / or determine the amount of the particular lanthanide or actinide. Methods for preparing a standard curve are known in the art.

[0122] The detection and / or quantification method can be performed on a variety of samples. Non-limiting examples of samples include drinking water, wastewater, groundwater, ash ponds, aqueous extracts from contaminated soil, wastewater (e.g., mine drainage such as acid mine drainage), or leachate (e.g., e-waste leachate or ore leachate). In various other examples, the sample is a solid sample. The method can be applied to samples with various pH values. For example, the sample has a pH of 6 or less (e.g., 5.5 or less, 5 or less, 4.5 or less, 4 or less, 3.5 or less, or 3 or less). In various examples, the pH is greater than 6.

[0123] Various lanthanides (e.g., lanthanide ions) and / or actinides (e.g., actinide ions) may be bound by the protein and / or device. For example, the lanthanide is selected from Tb, Eu, Dy, Sm, Nd, Yb, and ions thereof. In various examples, the lanthanide is Tb or its ion. The bound lanthanides and / or actinides may be the same or different. The concentration of the lanthanide and / or actinide in the sample may be less than 1 ppm.

[0124] The following description provides various examples and embodiments of the present disclosure. Description 1 A protein capable of binding metals and / or metal ions (e.g., in various examples, the protein can dimerize upon contact with a metal and / or metal ion), comprising a first EF hand motif, a second EF hand motif, a third EF hand motif, and a fourth EF hand motif, each comprising 11, 12, or 14 amino acid residues, wherein when the first EF hand motif, the second EF hand motif, the third EF hand motif, and the fourth EF hand motif each have 12 amino acid residues, each EF hand motif has the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E where: i) First EF hand motif, second EF hand motif, and fourth EF hand motif each X 1 are independently D or N; each X 2 are independently any canonical amino acid; each X 3 is independently D, N, or E; each X 4 are independently any canonical amino acid; each X 5 is independently D, N, or E; each X 6 are independently any canonical amino acid; each X 7 are independently any canonical amino acid; each X 8 are independently hydrophobic residues; each X 9 is independently D, E, or T; each X 10 are independently hydrophobic residues; and each X 11 are independently any canonical amino acid. ii) Regarding the third EF hand motif X 1 is N; X 2 is any canonical amino acid; X 3 is D; X 4 is G or A; X 5 is D or N; X 6 is any canonical amino acid; X 7 is T or S; X 8 is a hydrophobic residue; X 9 is E; X 10 is a hydrophobic residue; and X 11 is D; and iii) The EF hand motifs are linked by a linker of 12 or 13 amino acid residues, each amino acid residue of the linker being a canonical amino acid, except that the third and fourth EF hand motifs are linked by the following sequence: (X)-R-(X) (where each X is independently a canonical amino acid, and at least one amino acid of either linker is hydrophobic). Statement 2 X in the first EF hand motif, the second EF hand motif, and / or the fourth EF hand motif 7 is independently T or S. Statement 3 X in the third EF hand motif 4 A protein according to statement 1 or statement 2, wherein Statement 4 X in the third EF hand motif 8 is L. Statement 5 X in the third EF hand motif 10 is L, I, or M. Statement 6 A protein with enhanced REE / FREE selectivity, comprising a first EF hand motif, a second EF hand motif, a third EF hand motif, and a fourth EF hand motif, each EF hand motif comprising 11, 12, or 14 amino acid residues, wherein when the first EF hand motif, the second EF hand motif, the third EF hand motif, and the fourth EF hand motif each have 12 amino acid residues, each EF hand motif has the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E where: i) First and fourth EF hand motifs each X 1 are independently D or N; each X 2 are independently any canonical amino acid; each X 3 are independently D, N, or E; each X 4 are independently any canonical amino acid; each X 5 are independently D, N, or E; each X 6 are independently any canonical amino acid; each X 7are independently any canonical amino acid; each X 8 are independently hydrophobic residues; each X 9 are independently D, E, or T; each X 10 are independently hydrophobic residues; and each X 11 are independently any canonical amino acid; ii) Regarding the second EF hand motif X 1 is N; X 2 is any canonical amino acid; X 3 is D; X 4 is any canonical amino acid; X 5 is D; X 6 is any canonical amino acid; X 7 is T or S; X 8 is a hydrophobic residue; X 9 is E; X 10 is any canonical amino acid; and X 11 is any canonical amino acid; and iii) Regarding the third EF hand motif X 1 is D; X 2 is any canonical amino acid; X 3 is D; X 4 is D; X 5 is D; X 6 is G; X 7 is T or S; X 8is a hydrophobic residue; X 9 is D; X 10 is any canonical amino acid; and X 11 is any canonical amino acid; iv) at least one X in the second EF-hand motif and at least one X in the third EF-hand motif 2 is P; and v) The EF hand motifs are linked by linkers of 12 or 13 amino acid residues, where each amino acid in the linker is a canonical amino acid and at least one amino acid in either linker is hydrophobic. Statement 7 X in the second EF hand motif and / or the third EF hand motif 8 is L, I, M, or V. Statement 8 10. The protein of any one of the preceding statements, wherein the protein comprises the sequence: MKLSLKAGAA ITAFVFAASP VLAASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 1) or MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 2) or A protein having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to either SEQ ID NO: 1 or SEQ ID NO: 2, or comprising the following sequence: MLTGKEFLRKYNKDKDSTVEIVEAIDLGTKVFKAINPDKDKTLEAAETKGRLSDEDWAQFNKDGDKTLELDEWLIIVRKRFNDADANKDGKLTEAELDAPAGQQLILLIAK (SEQ ID NO: 7), or A protein with 70% identity to the sequence. Statement 9 10. The protein of any one of the preceding statements, wherein the protein is complexed with a rare earth element. Statement 10 10. The protein of statement 9, wherein the rare earth element is a light rare earth element. Description 11 10. The protein of statement 9, wherein the rare earth element is a heavy rare earth element. Statement 12 10. The protein of any one of the preceding statements, wherein the protein comprises the sequence: MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILX TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 44) where X is any canonical amino acid residue other than R. Statement 13 13. The protein of statement 12, wherein the protein comprises the sequence: MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILK TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 4). Statement 14 13. The protein of any one of statements 1 to 12, wherein the protein has the sequence: TIFF2025532059000011.tif222169TIFF2025532059000012.tif221169TIFF2025532059000013.tif22616 9TIFF2025532059000014.tif230169TIFF2025532059000015.tif237169TIFF2025532059000016.tif45169 Statement 15 13. The protein of any one of statements 1 to 12, wherein the protein has the sequence: MKLSLKAGAA ITAFVFAASP VLAASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 1); MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 2); or MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILK TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 4). Statement 16 A device comprising a protein according to any one of the preceding statements. Statement 17 17. The device of statement 16, wherein the device is a filter, a membrane, a sensor, a portable detector, a plate reader, a fluorometer, a biosensor, or an in-line monitor. Statement 18 A kit comprising a protein according to any one of statements 1 to 15, or a device comprising a protein according to any one of statements 1 to 15. Statement 19 16. A method for isolating a rare earth element, the method comprising contacting a protein according to any one of statements 1 to 15 with a sample containing the rare earth element, wherein the rare earth element binds to one or more proteins according to any one of statements 1 to 15, thereby removing the protein from the sample. Description 20 20. The method of claim 19, wherein the sample is drinking water, wastewater, groundwater, ash pond, aqueous extract from contaminated soil, wastewater, leachate, aqueous extract or leachate from solid waste such as electronic waste, aqueous extract or leachate from ore or mine tailings, or a solid sample. Statement 21 21. The method of claim 19 or 20, wherein the one or more rare earth elements are lanthanides selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y, and ions thereof. Statement 22 22. The method of any one of statements 19-21, wherein the method further comprises detecting and quantifying one or more rare earth elements. Statement 23 23. The method of any one of statements 19 to 22, wherein a plurality of different rare earth elements are bound to the protein. Statement 24 24. The method of statement 23, wherein each different rare earth element is individually separated from the protein. Description 25 16. A method for determining whether a light rare earth element is present in a sample, the method comprising contacting the sample with a protein according to any one of statements 1 to 15, and determining whether the protein has formed a dimer.

[0125] The following examples provide various illustrations of the present disclosure and are not intended to be limiting in any way. Example 1

[0126] This example describes the use of the peptides / proteins of the present disclosure.

[0127] Technologically important rare earth elements are notoriously difficult to separate due to subtle differences in their ionic radii and coordination numbers. Lanmodulin, a natural lanthanide-binding protein, offers a sustainable alternative to traditional solvent extraction-based separations. Herein, we characterized a novel lanmodulin (Hans-LanM) from Hansschlegelia quercus (Hans-LanM) with an oligomeric state sensitive to the rare earth ionic radius (the lanthanum(III)-derived dimer is more than 100 times tighter than the dysprosium(III)-derived dimer). The X-ray crystal structure showed how the picometer-scale difference in radius between lanthanum(III) and dysprosium(III) is transmitted to the quaternary structure of Hans-LanM by a carboxylate shift, which rearranges the second-sphere hydrogen-bonding network. Comparison with the prototype lanmodulin from Methylorubrum extorquens reveals distinct metal coordination strategies, which theoretically explain the higher selectivity of Hans-LanM for rare earths. Finally, structure-guided mutagenesis of key residues at the dimer interface of Hans-LanM tunes its dimerization in solution, enabling a single-step, column-based separation of neodymium(III) / dysprosium(III) mixtures (purity of each element >98%). This disclosure demonstrates the natural diversity of selective lanthanide recognition motifs and identifies rare-earth-sensitive dimerization as a biological principle for tuning the performance of biomolecule-based separation processes.

[0128] Here, we describe that ranmodulin from the methylotrophic bacterium Hansschlegelia quercus (Hans-LanM), isolated from English oak buds, exhibits enhanced RE resolution compared to Mex-LanM. While Mex-LanM is always monomeric, Hans-LanM exists in a monomer / dimer equilibrium state, the position of which depends on the specific RE bound to it. Three X-ray crystal structures and structure-guided mutagenesis of ranmodulin demonstrate the RE-dependent oligomeric state of Hans-LanM and its greater resolution compared to Mex-LanM. We also describe the one-step resolution of the critical neodymium / dysprosium pair using Hans-LanM. These results demonstrate a method for improving RE resolution by exploiting intermolecular interactions that are common in proteins but rare in small molecules.

[0129] Distinct selectivity profile of Hans-LanM Several features of Lanmodulin have been proposed. First, the LanM protein contains four EF-hand motifs. The EF-hands contain 12 residues and are flanked by a carboxylate-rich metal-binding loop and an α-helix, which are conventionally thought to be involved in Ca binding. II Although EF-hands 1-3 were responsive to binding (in Mex-LanM), they bound lanthanide(III) ions with low picomolar affinity and Ca II 10 for 8 EF4 binds with only micromolar affinity. Second, the adjacent EF-hand in LanM binds Ca. IIInstead of the typical ~25 residues of β-responsive EF-hand proteins, the metal-binding sites are separated by 12–13 residues, resulting in an unusual triple-helical bundle structure with the metal-binding site located at the periphery. Third, at least one EF-hand contains a proline at position 2 (in Mex-LanM, all four EF-hands contain the P2 residue). Database searches using the first two criteria and a sequence length of less than 200 residues identified 696 putative LanMs. These sequences were visualized using a sequence similarity network to identify LanM sequences that cluster separately from Mex-LanM. At a 65% identity threshold, a small cluster of sequences formed, separate from the main cluster (consisting of 642 sequences) (Fig. 1a). This exclusive cluster (the "Hans cluster") included several genera of bacteria, including Hansschlegelia and Xanthobacter (Fig. 50), all of which are facultative methylotrophs.

[0130] Hans-LanM shares low (33%) sequence identity with Mex-LanM (Fig. 5) and features a distinct EF-hand motif, particularly at positions 1, 2, and 9, which are critical in Mex-LanM and other EF-hand proteins (Fig. 1b). Hans-LanM therefore provided an opportunity to determine the features essential for selective lanthanide recognition in ranmodulin.

[0131] Hans-LanM was expressed in Escherichia coli as a 110-amino acid protein (Figure 5). III and Nd III is a representative LRE, and Dy III was selected as a representative HRE. ICP-MS revealed that this protein, like Mex-LanM, contains approximately 3 equivalents of La. III and Nd III binds to slightly less Dy IIIFurthermore, similar to Mex-LanM, Hans-LanM exhibits little helical content in the absence of metal, as judged by the circular dichroism (CD) signal at 222 nm (Fig. 1c). Unexpectedly, La III or Dy III Only two equivalents of are sufficient to induce a complete conformational change in Hans-LanM (Figure 6), indicating that a third binding equivalent is weak and does not increase helicity.

[0132] The apparent dissociation constant (K d,app ) は , reflecting the RE / RE selectivity and RE / non-RE selectivity of Mex-LanM under competitive RE recovery conditions. d,app When the determination of La (controlling the free metal concentration by a competitive chelator) was applied to Hans-LanM, the results (Fig. 1d, Table 2) differed from those of Mex-LanM. III and Nd III Binding of α to Hans-LanM increased the molar ellipticity at 222 nm by 2.3-fold, and a complete conformational change was revealed by stoichiometric titration. This conformational change was cooperative (Hill coefficient n = 2, Table 2), and K d,app The values ​​were similar, 68 pM and 91 pM, respectively. III In stoichiometric titration, La III Although it induces the same overall response as the chelator buffered Dy (Figure 6), III In the titration, Hans-LanM showed a smaller conformational response (1.8-fold increase). This difference was due to the Dy III This suggests that at least one of the binding sites exhibits very weak reactivity (K d,app >0.3 μM, the highest concentration available in a chelator buffer titration). III The main response to Y occurs at 2.6 nM, >30-fold higher than the LRE, with little to no cooperativity (n = 1.3). In contrast, Mex-LanM shows only a slight preference for the LRE (approximately 5-fold, Figure 1e), and exhibits a strong preference for all lanthanides and Y. IIIHans-LanM reacts weakly with calcium(II) (K d,app = 60 μM), Dy III The Hans-LanM complex showed a similar lack of cooperativity (n = 1.0) and partial structural changes (Figure 51). Thus, Hans-LanM discriminates between LRE and HRE more strongly than Mex-LanM (the HRE complex exhibits lower affinity, less cooperativity, and less primary structural changes).

[0133] LRE-selective dimerization The different behavior of the LRE- and HRE-Hans-LanM complexes suggested a mechanism for LRE / HRE selectivity that is not present in Mex-LanM. Because Mex-LanM is similarly monomeric in complexes with LREs and HREs, it was suggested that the LREs and HREs may induce different oligomeric states in Hans-LanM. III In the presence of Nd, Hans-LanM eluted from a size-exclusion chromatography (SEC) column at 27.8 kDa instead of the expected molecular weight (MW) of 11.9 kDa, suggesting a dimer (Figures 7 and 8a). III Gradually, but Gd III The apparent MW rapidly decreased toward the value expected for a monomer (Fig. 2a, Fig. 8, Table 3). III Heavier lanthanides do not appear to support the growth of RE-utilizing bacteria.

[0134] To further support preferential dimerization in the presence of physiologically relevant LREs, we analyzed the Hans-LanM RE complex using multi-angle light scattering (MALS) (Figure 2a, Figure 9). III , Nd III and Gd III The MW of the complex is 22-25 kDa, indicating a dimer. III Decreased from Dy III and Ho III (approximately 15 kDa) (Table 17), consistent with the SEC data. IIThe bound Hans-LanM also showed a MW of 14.7 kDa. II The apo- and apo-Hans-LanM complexes are three times larger than expected for the monomer, suggesting that these forms exist in rapid equilibrium under these conditions with a monomer:dimer ratio of approximately 2:1. III -bond type, and Dy III -bound Hans-LanM dimerization K d (K dimer ) was measured by isothermal titration calorimetry (ITC) (Figures 10-12, Table 18). III The binding protein dimerizes weakly, and K dimer The values ​​of La were 117 μM and 60 μM, respectively, consistent with the ratio of monomer to dimer reflected by the SEC and MALS traces. III In the presence of K, the dimer is too tight to observe monomerization by ITC. dimer The results showed that the La III is Dy III This favors Hans-LanM dimerization by more than 100 times.

[0135] La III The 1.8 Å resolution X-ray crystal structure of Hans-LanM complexed with LanM confirms LRE-induced dimerization (Figure 52, Table 4). Two LanM monomers interact head-to-tail (Figure 2b), separating them by approximately 600 Å through hydrophobic and polar contacts. 2 The dimer interface buries the surface area of ​​the dimer (Fig. 2c-d). These interactions occur primarily between side chains contributed by core helices α2 (between EF1 and EF2) and α3 (between EF3 and EF4) (Fig. 13). Residues at the dimer interface are in direct contact with only one of the four metal-binding sites (EF3), and three residues of EF3 from each monomer form a hydrogen-bonding network with Arg100 from the other monomer (Fig. 2c), suggesting that the occupancy and coordination geometry of this site may control the oligomeric state.

[0136] Hans-LanM and its complex (3 equivalents of La III , Nd III , Dy III The solvent envelope calculated from the SAXS data is shown in Figures 14-15. III -Hans-LanM fits well to the crystallographic Hans-LanM dimer, and Nd III -Hans-LanM was a good fit, but Dy III -Hans-LanM did not provide a good fit (Fig. 2e, Fig. 16-18). III The weak dimerization of Hans-LanM is also supported by quantitative metrics such as Porod volume (Tables 5-6, Figures 19-20). Taken together, the biochemical and structural results indicate that the dimerization equilibrium of Hans-LanM is strongly dependent on the specific RE bound.

[0137] Structural basis of dimerization Furthermore, La III The structure of Mex-Hans-LanM provides the first detailed view of the coordination environment in lanmodulin, and indeed any natural biomolecule responsible for reversible lanthanide recognition. The previous NMR structure of Mex-LanM revealed an unusual protein folding but failed to provide molecular-level details of the metal binding site. To understand the basis of LRE / HRE discrimination, we investigated the Dy III The 1.4 Å resolution structure of Nd-Hans-LanM was determined. III A 1.01 Å resolution structure of -Mex-LanM is reported, rationalizing the shallower RE selectivity trend of Mex-LanM.

[0138] La III -In Hans-LanM, EF1-3 is La III ions (Figure 52b-e). EF4 is structurally different, being occupied by La III showed no abnormal difference density consistent with Na I (Fig. 21a). IIIThe binding site is ten-coordinated, as observed in the structure of lanthanide-dependent methanol dehydrogenase (Figure 22). It contains a monodentate Asn (N1 position), four bidentate Glu / Asp residues (D3, D5, E9, E 12 ), and the backbone carbonyl (T7 / S7) constitute the first coordination sphere of EF1-3 (Fig. 3a). No extraneous solvent ligands were observed (Fig. 21b), and Eu III The number of coordinated solvent molecules (q) was determined by Hans-LanM luminescence studies, which gave a result of q = 0.11, consistent with the absence of solvent ligands in the X-ray structure (Figure 23).

[0139] The lanthanide-binding sites of Hans-LanM share more extensive second-sphere interactions, which may further constrain the position of the ligands and the size of the metal-binding cavity (Figure 24). This phenomenon is most evident in EF3, where the dimer interface mediates an extended hydrogen-bonding network involving multiple ligands. Arg100 is contributed from the neighboring monomer and protrudes into the solvent-exposed side of EF3, contacting two carboxyl group ligands, Asp85 (D3) and Glu91 (E9), reinforcing its bidentate binding mode. Arg100 also contacts Asp93 (EF3 D3). 11 ), which is specific to EF3 in Hans-LanM and not observed in Mex-LanM. The importance of this network for the dimerization of Hans-LanM was verified by creating the minimal mutation, R100K. R100K-Hans-LanM had almost the same K as wild-type Hans-LanM. d,app The value indicates Nd III and Dy III The responses to La III K d,app was twice as weak (Table 7, Figure 25). SEC-MALS analysis revealed that apo-, La III -, and Dy III The MW of -R100K-Hans-LanM was 10-13 kDa (Figure 26, Table 8), and La III The complex showed increased monomerization, and La IIIThe low affinity of Arg100-EF3 may be due to weak dimerization. All four residues that make up the Arg100-EF3 network are completely conserved in the Hans cluster (Fig. 27), suggesting that these interactions may contribute to dimerization in these ranmodulins.

[0140] Dy III The structure of Dy-Hans-LanM confirms the importance of second-sphere control of ligand position (Figure 53, Figures 28-30, Tables 9-10). III The overall structure of -Hans-LanM is La III -Hans-LanM almost overlaps, and EF1-3 Dy III The coordination sphere of the ion is La III The coordination sphere of La-Hans-LanM is similar (Fig. 3a inset), with the notable exception of E9 (e.g., Glu91 in EF3). III From bidentate coordination with ions, smaller Dy III The low coordination number with the HRE ion is consistent with other RE complexes. In EF3, this carboxylate shift results in a distance between Arg100 and the proximal Oε of Glu91 of 2.9 Å (La III -Hans-LanM) to 3.2 Å (Figure 31). This rearrangement of the second-sphere hydrogen bond network dimer This suggests a structural basis for the RE-dependent differences in values.

[0141] The metal binding site of Mex-LanM is substantially different from that of Hans-LanM. In Mex-LanM, all four EF hands bind Nd with either 9-coordinate (EF1-3) or 10-coordinate (EF4) Nd. IIIThe metal sites are occupied by ions, each containing two solvent ligands not present in Hans-LanM (Fig. 3b, Fig. 32). The observation of two solvent molecules per metal site and hydrogen bonding to the D9 residue validates recent spectroscopic studies. The difference in coordination number between EF1-3 and EF4 is due to the D3 carboxylate being monodentate in EF1-3 but bidentate in EF4. The Nd in Mex-LanM III The part is Dy III / La III It shares the nine-coordinate / ten-coordinate structure observed in -Hans-LanM but the seven-coordinate Ca of calmodulin. II The increased coordination number of Mex-LanM for calmodulin is due to the bidentate D5 and additional solvent ligands. These similarities lead to a unique 10-fold binding site for the RE of LanM. 8 times (Ca II These results suggest that much of the selectivity (vs. LRE / HRE) of Hans-LanM stems from subtle differences in the second coordination sphere and other more distal interactions. Finally, the exclusively protein-derived first coordination sphere in Hans-LanM, particularly due to coordination by E9, forms a more extended hydrogen-bonding network (Figures 24 and 33), likely providing enhanced control over the radius of the binding site. Thus, this structure rationalizes the extraordinary RE / non-RE selectivity of Mex-LanM and Hans-LanM, as well as the difference in LRE / HRE selectivity.

[0142] Nd III / Dy III One-step separation of The differences in stability and structure of the LRE vs. HRE complexes of Hans-LanM suggest that Hans-LanM (wild-type and / or R100K) is superior to Mex-LanM in RE / RE separation. III and Dy IIIWe focused on isolating the RE pair. First, we analyzed the stability of wild-type and R100K-Hans-LanM RE complexes with citrate (previously used as a release agent for Mex-LanM). RE-Hans-LanM complexes were generally less stable with citrate than Mex-LanM, as expected from their lower affinity (Fig. 1e), but Nd III -Hans-LanM complex and Dy III The difference in stability between the Hans-LanM complex and the citrate concentration required for 50% desorption of each metal ([citrate] 1 / 2 ) and reported by the fluorescence of the two Trp residues of Hans-LanM (Fig. 34), is twice as high as that of the Mex-LanM complex (Fig. 4a, Table 11, Fig. 54). Furthermore, the R100K mutation significantly reduced the La III The complex is significantly destabilized by citrate, but Nd III The complex is only slightly affected, and Dy III This result suggests that dimerization does not affect the LRE complex of Hans-LanM (especially La III The R100K mutation selectively stabilizes the Dy complex, confirming that this is the factor lost by the R100K mutation. III is easily detached from both Hans-LanM and R100K at 10-100mM of the chelating agent, but Nd III Since there is no significant desorption, Nd III / Dy III The conditions for separation are suggested (Fig. 4b).

[0143] Although the two-fold modulation of RE / RE selectivity by dimerization may seem small, such differences offer an opportunity to reduce the number of separation stages and increase the efficiency of the separation process. Therefore, we immobilized the Hans-LanM and R100K variants via their C-terminal Cys residues on maleimide-functionalized agarose beads (as previously reported) and analyzed Nd III / Dy IIISeparation studies were performed. Immobilized Hans-LanM bound approximately 1 equivalent of RE, compared with 2 equivalents for Mex-LanM and R100K-Hans-LanM (Figure 35). Hans-LanM and R100K showed similar resolution in the La-Gd range (although R100K showed higher Gd-Dy resolution), as determined by the on-column partition ratios (D) of mixed RE solutions at equilibrium (Tables 12–14, 19, and 19c). These Nd / Dy separation factors were approximately 2-fold (Hans-LanM) and 3-fold (R100K-Hans-LanM) of Mex-LanM (Table 19). Immobilized Hans-LanM was loaded to 90% of its breakthrough capacity (using a model e-waste mixture consisting of 5% dysprosium and 95% neodymium) and eluted with a short stepwise malonate gradient, as shown in Figure 4b, followed by complete desorption with HCl at pH 1.5. In a single purification step, Dy was upgraded from 5% to 83% purity, and Nd was recovered at 99.8% purity (both >98% yield) (Figure 5). This significantly outperformed a comparable process based on Mex-LanM, which achieved only 50% purity in the first separation step and required a second step to achieve >98% purity. The immobilized R100K variant showed even better performance, eluting Dy in a single step. III and Nd III We achieved baseline separation of the Hans-LanM dimers (>98% purity and >99% yield) (Figure 4d). The better performance of the R100K variant was unexpected and may suggest that functional dimers are unlikely to exist on the column at this immobilization density (see Figure 55 for discussion). Thus, despite the significant improvement in performance compared to Mex-LanM, characterization of the dimerization mechanism of Hans-LanM suggests that fully utilizing the dimerization phenomenon on the column may require linking two monomers onto a single polypeptide chain, which is currently under investigation.

[0144] conclusion Biochemical and structural characterization of the metal-sensitive dimerization mechanism of Hans-LanM provides a novel allosteric mechanism for LRE / HRE selectivity in biology, extending the concept of dimerization-dependent metal recognition recently discovered in synthetic lanthanide complexes and artificial transition metal-binding proteins, demonstrating that these principles are deeply rooted in nature. This disclosure demonstrates that dimerization strength, and therefore metal selectivity, can be rationally tuned. Hans-LanM evolved LRE-selective dimerization at physiological protein concentrations closer to the concentrations used in the biochemical assays disclosed herein (10-20 μM), rather than the on-column concentrations (approximately 3 mM). Therefore, to utilize dimerization in separation processes, it is useful to shift the sensitivity of dimerization to higher concentrations, for example by adjusting hydrophobic interactions at the dimerization interface. Furthermore, these studies establish that ranmodulins with as little as 33% identity have useful differences in metal selectivity. Finally, the solvent-excluded coordination sphere of Hans-LanM should outperform Mex-LanM in RE / actinide separation, luminescence-based sensing, and stabilization of hydrolysis-prone ions. Continued characterization of the coordination and supramolecular principles of biological element recognition will facilitate the design of ligands with higher RE / RE selectivity and their implementation in novel RE separation processes.

[0145] Bioinformatics Methods a) Protein and genome sequence data Using the LanM sequence of M. extorquens AM1 as a query, PSI-BLAST searches were performed against the NCBI (National Center for Biotechnology Information) nonredundant protein sequence (nr) and metagenomic protein (env_nr) databases until convergence was achieved. The resulting 3,047 protein sequences were then manually curated for sequences less than 200 residues in length, with at least one EF-hand pair separated by less than 14 residues, and containing four EF-hands. The signal peptide of the LanM sequence was predicted using SignalP (v6.0) and removed before further sequence analysis. b) Construction of sequence similarity networks Using the Enzyme Function Initiative-Enzyme Similarity Tool, we set the E-value threshold at 1 × 10 -5 The similarity between all peptide sequence pairs was calculated as follows. A sequence similarity network (SSN) consisting of 696 nodes and 241,853 edges was then constructed and explored using organic layout in Cytoscape (v3.9.1) and visualized in R (v4.1.0). The edge identity threshold was gradually increased from 40% to 90%, resulting in clear clusters. c) Multiple sequence alignment and phylogenetic analysis LanM sequences were aligned using MUSCLE (v5.1) with default parameters. The model used to construct the phylogeny was selected using ModelFinder in IQ-TREE (v2.2.0.3) (-mset set to beast2). Based on these results, a Bayesian phylogeny was created using BEAST (v2.6.7). The resulting phylogeny was then scaled to 10. 7 The results were evaluated over generations, discarding a 25% burn-in, and visualized using ggtree (v3.2.1).

[0146] Expression and purification of Hans-LanM and its R100K variant The gene encoding Hans-LanM (codon-optimized for expression in E. coli and with the original 23-residue signal peptide removed) (see Table 15) was obtained from Twist Bioscience and inserted into pET-29b(+) using the NdeI / XhoI restriction enzyme sites. Hans-LanM was overexpressed at a 2-L scale and purified using the protocol established for Mex-LanM, with one modification: after a final size-exclusion chromatography (SEC) step, the protein was concentrated to 5 mL and dialyzed against 5 g of Chelex-100 (in 500 mL of 30 mM HEPES, 100 mM KCl, 5% glycerol, pH 8.4) and purified with Ca. IIThis procedure yielded approximately 15 mL of 550 μM protein, which was not further concentrated. The final yield was 45 mg of protein per liter of culture. Protein concentration was calculated as 11,000 μM based on the ExPASy ProtParam tool. -1 cm -1 The extinction coefficient was calculated using the same procedure. R100K-Hans-LanM was also purified using the same procedure, yielding 30 mg of protein per 1 L of culture.

[0147] Circular dichroism (CD) spectroscopy CD spectra of Hans-LanM were collected at 15 μM (monomer concentration) in Chelexed Buffer A (20 mM acetate, 100 mM KCl, pH 5.0) as described unless otherwise noted. Buffered metal solutions were prepared as described. Other details are described herein.

[0148] Preparation of protein samples for SEC-MALS and SAXS Samples of wild-type Hans-LanM were prepared by slowly adding 3.0 equivalents of metal (0.5 equivalents each) to 1.0 mL of a concentrated stock (550 μM) of Hans-LanM. At these protein concentrations, the LRE samples (e.g., La III ), slight precipitation was observed in the HRE samples (e.g., Dy III ), significant precipitation was observed. The sample was centrifuged at 12,000 × g for 2 min to remove the precipitate, and then purified using gel filtration chromatography (HiLoad 10 / 300 Superdex 75 pg, 1 mL loop, 0.8 mL / min) in buffer B (30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0). The peak containing Hans-LanM (elution volume range 12.0–15.0 mL) was collected, avoiding the high-MW aggregate peak, to yield 2.0 mL of metallated Hans-LanM at 114 μM–128 μM (1.37–1.53 mg / mL).

[0149] Samples of R100K-Hans-LanM do not form or precipitate high molecular weight species upon metal addition. To prepare samples of this protein, a 500 μM protein solution was diluted to 250 μM (3 mg / mL) with buffer B containing 0.75 mM specific RECl3, yielding a final solution of 3 mg / mL protein at a 1:3 metal ratio, which was directly analyzed by SEC-MALS.

[0150] For calcium conditions, the protein was diluted to 250 μM (3 mg / mL), 5 mM CaCl2 was added, and the sample was incubated at room temperature for 1 h. The buffer used for SEC-MALS was the same as above, except that it contained 5 mM CaCl2.

[0151] In-line size exclusion chromatography and multi-angle light scattering (SEC-MALS) SEC-MALS experiments were performed using an Agilent 1260 Infinity II HPLC system equipped with an autosampler and fraction collector and a Wyatt SEC hydrophilic column (5 μm silica beads, 100 Å pore size, dimensions 7.8 × 300 mm). A Wyatt Technology DAWN MALS and a Wyatt Optilab Refractive Index (RI) detector were used to analyze the molar mass of peaks eluted from the column. The SEC-MALS system was equilibrated with buffer B for 5 h. The system was calibrated using bovine serum albumin (BSA, monomer MW: 66 kDa) in the same buffer to normalize and align the MALS and RI detectors. 15 μL of each sample was injected at a flow rate of 0.8 mL / min, and the chromatographic run time was 25 min. Data were analyzed using ASTRA software (Wyatt). If SAXS analysis was required, a second run was performed by injecting 150 μL of protein (approximately 4 mg / mL) and collecting 200 μL fractions of the main peak. BioSAXS data were then collected in triplicate.

[0152] Isothermal titration calorimetry apo, La IIIBonds, and Dy III The dissociation constant of the bound Hans-LanM dimer was determined by diluting the concentrated protein stock and performing ITC (TA Instruments Low-volume Auto Affinity ITC). The syringe contained 300 μM protein (apo, or 2 equivalents of Dy). III binding) or 150 μM or 540 μM (2 equivalents of La III The cell contained 185 μL of matching buffer (30 mM MOPS, 100 mM KCl, pH 7.0). Titrations were performed at 30°C. Titrations consisted of an initial 0.2 μL injection followed by 17 × 2 μL injections unless otherwise noted, with stirring at 125 rpm and 180 seconds of equilibration time between injections. Heat quantities were fitted in NanoAnalyze using the "Dimer Dissociation" model to determine the dimer dissociation constant (K dimer ), dissociation enthalpy (ΔH), and dissociation entropy (ΔS) were obtained. All parameters are shown in Table 18.

[0153] K dimer is defined as the dissociation constant of the following equilibrium TIFF2025532059000017.tif6169(K dimer =[M] 2 / [D]) where [D] is the concentration of dimer, [M] is the concentration of monomer, and the total protein concentration [P] (measured using the extinction coefficient of the monomer) is given by [P] = [M] + 2[D]. Therefore, TIFF2025532059000018.tif16169This formula is the K calculated from the ITC experiment. dimer Using the [P] value and [P] from the SEC-MALS trace, it can be used to estimate the monomer and dimer concentrations during a SEC-MALS experiment. This equation can be used to calculate the La concentration from the SEC-MALS data. III Maximum possible K of the binding protein dimer It can also be used to estimate

[0154] BioSAXS Small angle X-ray scattering (BioSAXS) was collected on RE-complexed Hans-LanM at the protein concentrations shown in Table 5 using the equipment and conditions described herein.

[0155] Forward scattering I(0) and radius of gyration (R g ) are listed in Table 5 and were calculated using the Guinier approximation. This is because the angle q is very small (q<1.3 / R g ), the intensity is I(q)=I(0)exp[-1 / 3(qR g ) 2 ] is assumed to be approximated by La III -, Nd III -, Dy III In the -bound condition, this was consistent with the calculated size of the crystallographic dimer of 17.9 Å. The molecular weight was estimated from comparison with SAXS data of a BSA standard. The data files were analyzed using ATSAS software in Guinier R g , maximum particle diameter (D max ), Guinier fits, Kratky plots, and pair-distance distribution functions. GNOM within ATSAS was used to calculate the pair-distance distribution function P(r), from which R g and D max was determined. The solvent envelope was calculated using DENSS. The theoretical scattering profile of the constructed model was calculated and fitted to the experimental scattering data using CRYSOL. The fractions of monomer and dimer were estimated using OLIGOMER.

[0156] Protein sample preparation for crystallography To 2 mL of Hans-LanM (1.16 mM, Buffer B), 3.0 equivalents of LaCl3 or DyCl3 were slowly added (0.5 equivalents each, mixed to minimize precipitation). The precipitate was removed by centrifugation at 12,000 × g for 2 minutes. Any soluble aggregates were removed, and the protein was exchanged into a buffer lacking glycerol (Buffer C: 30 mM MOPS, 50 mM KCl, pH 7.0) by gel filtration chromatography (HiLoad 16 / 600 Superdex 75 pg, 1 mL loop, 0.75 mL / min). The peak between 70 and 85 mL was pooled, and the fractions were concentrated to approximately 500 μL for a final concentration of approximately 1.3 mM.

[0157] Mex-LanM was purified as previously described and exchanged into buffer C before crystallization. III (NdCl3) was added.

[0158] Common crystallographic methods Diffraction data sets were collected at the Life Sciences Collaborative Access Team (LS-CAT) ID-G beamline and processed with the HKL2000 package. For all structures, phase information was obtained using the single-wavelength anomalous diffraction (SAD) method with lanthanide ions identified in HySS as anomalous scatterers using phenix.autosol. Initial models were generated using phenix.autobuild, followed by manual correction and refinement using Coot and phenix.refine. In the final stage of model refinement, the anisotropic displacement parameter (ADP) and occupancy were refined for all lanthanide sites. Model validation was performed on the Molprobity server. Figures were generated using the PyMOL molecular graphics software package (Schrodinger, LLC).

[0159] Structure determination of La-bound Hans-LanM Crystals were obtained using the sitting drop vapor diffusion method. 1 μL of protein solution (15 mg / mL) was mixed with 1 μL of 10 mM trisodium citrate (pH 7.0) and 27% (w / v) PEG 6000 at room temperature in a 24-well Hampton Research plate (catalog number HR1-002). Thin, plate-like crystals appeared within 3 days. Crystals suitable for data collection were mounted on rayon loops, briefly immersed in a cryoprotectant solution containing 10% ethylene glycol in the well solution, and flash-frozen in liquid N2.

[0160] La III Supported Hans-LanM was crystallized in the P21 space group (β = 90.024°) (four monomers in the asymmetric unit). The initial figure of merit (FOM) and Bayesian CC were 0.563 and 0.56, respectively. The final model consisted of residues 24-133 of each chain, 12 La III ions (3 per chain of the first, second, and third EF-hands), 4 Na I ions (one per chain of the fourth EF-hand), 273 water molecules, and two citrate molecules. As shown by Ramachandran statistical analysis, 100% of the modeled residues are in the allowed or preferred regions.

[0161] Structure determination of Dy-bound Hans-LanM Crystals were obtained using the sitting-drop vapor diffusion method. In this method, 1 μL of protein solution (15 mg / mL) was mixed with 1 μL of 250 μM trisodium citrate (pH 7.0) and 27% (w / v) PEG 6000 at room temperature in a 24-well Hampton Research plate (catalog no. HR1-002). Thin, plate-like crystals appeared within one month. Crystals suitable for data collection were mounted on rayon loops, briefly immersed in a cryoprotectant solution consisting of well solution plus Hampton Research perfluoropolyether cryo-oil (catalog no. HR2-814), and flash-frozen in liquid N2.

[0162] Dy III Supported Hans-LanM was crystallized in the P21 space group (β = 93.567°) (four monomers in the asymmetric unit). The initial FOM and Bayesian CC were 0.748 and 0.58, respectively. The final model consisted of residues 24-133 of each chain (except for chain D, where residues 34-38 could not be modeled), 14 Dy III ions (four in strands A and D, and three in the second, third, and fourth EF-hands of strands B and C), and 656 water molecules. Of the modeled residues, 100% are in the allowed or preferred regions, as shown by Ramachandran statistical analysis. The collection of the anomaly dataset is described herein.

[0163] Structure determination of Nd-bound Mex-LanM Crystals were obtained using the sitting-drop vapor diffusion method. In this method, 1 μL of protein solution (35 mg / mL) was mixed with 1 μL of 0.1 M ammonium sulfate, 0.1 M Tris pH 7.5, and 20% (w / v) PEG 1500 at room temperature in a 24-well Hampton Research plate (catalog no. HR1-002). Thin, plate-like crystals appeared within 6 months. Crystals suitable for data collection were mounted on rayon loops, briefly immersed in a cryoprotectant solution consisting of well solution plus Hampton Research perfluoropolyether cryo-oil (catalog no. HR2-814), and flash-frozen in liquid N2.

[0164] Nd III Supported Mex-LanM was crystallized in the P212121 space group (one monomer in ASU). The initial FOM and Bayesian CC were 0.799 and 0.56, respectively. The final model consisted of residues 29-133, four Nd III ions, and 171 water molecules. As shown by Ramachandran statistical analysis, 100% of the modeled residues are in the allowed or preferred regions.

[0165] Fluorescence spectroscopy All fluorescence data were collected using a Fluorolog-QM fluorometer (Horiba Scientific) in a 75-21-C configuration equipped with a double monochromator in the excitation arm and a single monochromator in the emission arm. A 75W xenon lamp was used as the light source for steady-state measurements, and a pulsed xenon lamp was used for time-resolved measurements. A 10mm quartz spectrofluorimeter cuvette (Starna Cells, 18F-Q-10-GL14-S) was used to collect data at 90° to the excitation beam path.

[0166] Fluorescence lifetime measurements were performed according to established methods. III A solution of Hans-LanM (4.5 mL total) containing 2 equivalents of α-Han-LanM was prepared in a 100% HO matrix (buffer: 25 mM HEPES, 75 mM KCl, pH 7.0). Half of this initial protein mixture (2.25 mL) was kept for future use, while the remainder was lyophilized to remove the HO and exchanged with DO by resuspending twice in 99.9% DO. The resulting protein solutions (in 100% HO and approximately 99% DO) were mixed in varying ratios to achieve DO contents of 0%, 25%, 50%, and 75%. The protein concentration was 20 μM. For each sample, the luminescence decay time constant (τ) was measured over 5000 shots over a 2500 μs time span (λ). ex = 394 nm, λ em= 615 nm). τ was determined using a single exponential fit with FelixFL Powerfit-10 software (Horiba Scientific). 1 / τ was plotted against the percent composition of DO, and the slope (m) of the resulting line was determined. q values ​​were determined using the following equation (Equation 2): TIFF2025532059000019.tif9169where τ -1 H2O and τ -1 D2O are the reciprocals of the time constants for 100% H2O and D2O, respectively (the latter extrapolated using the equation of the fitted line), and are expressed in ms -1 Also, n OH =0, n NH =0, n O-CNH = 1 (derived from the metal-coordinated Asn residue) (based on the Hans-LanM crystal structure). This equation simplifies to Equation 3: TIFF2025532059000020.tif9169

[0167] For fluorescence competition experiments, a 20 μM solution of Hans-LanM or R100K variant was prepared in buffer A (pH 5.0) containing 2 equivalents of metal (40 μM). Fluorescence emission spectra were measured at λ ex = 278 nm, λ em Spectra were collected at wavelengths 300-420 nm, integration time 0.5 s, and step size 1 nm. Titrations were performed by adding at least 0.6 μL of titrant (concentrated stock solutions of 10 mM to 1 M citrate or malonate, pH 5.0). Spectra were corrected for dilution. Each experiment was performed in triplicate.

[0168] Purification of Cys-containing variants R100K-Hans-LanM-Cys was expressed and purified as described for Mex-LanM-Cys, with a final yield of 50 mg protein / L culture. For Hans-LanM-Cys, the protein was purified by incorporating the same modifications as described above into the Mex-LanM-Cys purification, except for the dialysis step. The SEC step was performed using reducing buffer (30 mM MOPS, 100 mM KCl, 5 mM TCEP, pH 7.0) containing 5 mM EDTA, and the protein was frozen under liquid N2 before immobilization.

[0169] Maleimide functionalization of agarose beads Maleimide functionalization of amine-functionalized agarose beads has been described previously.

[0170] Fixation of Hans-LanM and R100K variants Immobilization of R100K-Hans-LanM was performed using a thiol-maleimide conjugation reaction as previously described. For Hans-LanM, a final protein concentration of approximately 0.4 mM (8 mL) was combined with 1 mL of maleimide microbeads, and the conjugation reaction was carried out at room temperature for 16 h. Unconjugated Hans-LanM was removed by washing with coupling buffer, and the Hans-LanM microbeads were stored in coupling buffer for subsequent testing. To quantify the immobilization yield of Hans-LanM, Pierce TM The LanM concentration in the reaction mixture before and after the conjugation reaction was measured using a BCA Protein Assay (ThermoFisher Scientific) as previously described.

[0171] Batch experiments to determine separation factors The LanM-immobilized microbeads were washed with DI water. A feed solution (5 mL, equimolar RE La-Dy, total volume 3 mM, pH 5.0) was added to 1 mL of microbeads and incubated for 2 h. The equilibrated liquid was collected, and the RE concentration was measured by ICP-MS in [M]. adNext, RE was desorbed from the microbeads using 4 mL of 0.1 M HCl, and the concentration was measured as [M]. de was measured by ICP-MS.

[0172] The RE partition coefficient (D) between the LanM phase and the solution phase was calculated as follows: TIFF2025532059000021.tif9169 where [M] LanM and [M] Liquid are the molar concentrations of each metal ion in the LanM phase and the solution phase at equilibrium, respectively. To account for free liquid adsorbed to the agarose microbeads, the following correction was applied: TIFF2025532059000022.tif7169

[0173] The separation factor (SF) is defined as: TIFF2025532059000023.tif8169 where, D RE1 and D RE2 are the partition coefficients of RE1 and RE2, respectively.

[0174] Breakthrough column experiment The columns were packed and analyzed for metal concentrations as described in our previous work, details of which are included herein.

[0175] In RE pair separation experiments, the purity and yield of metal ions are defined as follows: TIFF2025532059000024.tif15169 where C RE1 and C RE2 are the molar concentrations of RE1 and RE2, respectively.

[0176] Table 17. Characterization of Hans- and Mex-LanM metal complexes using SEC-MALS The concentrations of the protein samples loaded onto the column were as follows: 1.2-1.5 mg / mL for apo- and RE-bound Hans-LanM; 1.2-1.5 mg / mL for Ca; IIFor RE-conjugated Hans-LanM, 3 mg / mL; for Mex-LanM, 3 mg / mL. For RE-containing samples, the protein was diluted with 3 equivalents of the appropriate RE. III Preincubated with ions. II In the case of , 5 mM CaCl2 was added to the running buffer. The apoprotein eluted in two peaks, the first being a minor contribution (10% of the protein, 56.5 kDa) and the second being a major peak (90% of the protein, 12.9 kDa). For details on sample preparation, see Materials and Methods. Hans-LanM values ​​are plotted in Figure 2a. The raw data for La, Nd, and Dy are shown in Figure 9. JPEG2025532059000025.jpg108169

[0177] Table 18. ITC thermograms fitted to the dimer dissociation model for apo- and Dy III -Thermodynamic parameters of the Hans-LanM bond La III For the binding protein, these values ​​could not be determined because no change in the heat of measurement was observed during the titration experiments (Figure 12). Values ​​are reported as the mean with standard deviation of three independent titrations. TIFF2025532059000026.tif26169

[0178] Table 19. Distribution coefficients (D) and separation factors (SF) for equilibration of Nd / Dy binary solutions using Hans-Lan M or R100K-Hans-Lan M columns. The volumes of the Hans-LanM and R100K-Hans-LanM columns were 0.9 mL and 0.7 mL, respectively. The feed solution for this experiment was 5.0 mL and contained 1.42 (4) mM Nd and 1.62 (32) mM Dy, as determined by ICP-MS analysis. The pH was 5.0. This experiment confirms that the immobilized R100K variant exhibits superior on-column separation characteristics compared to wild-type Hans-LanM. See the legend in Table S12 for details on the uncertainty values ​​in parentheses. TIFF2025532059000027.tif59169

[0179] General considerations Chemical reagents were obtained from Millipore Sigma at the highest available purity unless otherwise noted. Chemically competent E. coli BL21(DE3) cells were obtained from NEB. Biochemical and column-based experiments were performed using RE chloride salts and buffers obtained from Millipore Sigma at ≥99.9% purity. Anion exchange chromatography was performed using Q Sepharose Fast Flow resin obtained from Millipore Sigma. Automated protein chromatography was performed on a GE Healthcare Biosciences Äkta Pure fast protein liquid chromatography (FPLC) system using a HiLoad Superdex 75 pg 16 / 600 column for preparative scale and a Superdex 75 pg Increase 10 / 300 GL column for analytical scale. RE was quantified using an inductively coupled plasma mass spectrometer (ICP-MS; Thermo Scientific iCAP RQ) in KED mode with He. The ICP-MS is located in the Laboratory for Isotopes and Metals in the Environment (LIME) at the Pennsylvania State University Earth and Environmental Systems Institute. For protein immobilization, amine-functionalized agarose beads were purchased from Nanocs. N-Succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was purchased from Chem-Impex International and used without further purification.

[0180] Circular dichroism (CD) spectroscopy CD spectra of Hans-LanM were collected as previously described. Specifically, samples were scanned from 195 to 255 nm using a Jasco J-1500 CD spectrometer with the following settings: 1 nm bandwidth, 0.5 nm data pitch, 50 nm / min scan rate, and 4 s averaging time. For all buffered metal and stoichiometric titrations, the cuvette contained 15 μM protein (monomer concentration). For stoichiometric titrations, the protein was diluted in Chelex-treated Buffer A (20 mM acetate, 100 mM KCl, pH 5.0) and titrated with 0.5–4.0 equivalents of each metal ion from a 1.5 mM solution in the same buffer.

[0181] Buffered metal solutions were prepared as previously described. II , La III , Nd III EDTA was used as a chelating agent for the titration of Dy III EGTA was used. Protein was added to "high" and "low" metal solutions of each metal ion up to 15 μM. These solutions were combined in various ratios to a final volume of 200 μL to create a range of free metal concentrations in the presence of Hans-LanM. These solutions were incubated overnight at 4°C. CD spectra were collected, and the CD signal at 222 nm was plotted against the free metal concentration, yielding a binding curve fitted using the Hill equation.

[0182] BioSAXS data collection Data were collected at the Penn State X-Ray Crystallography Facility home source at a wavelength of 1.54 Å and analyzed using BioSAXS2000 nanoX-rays were generated by a Rigaku MM007 rotating anode housed in a Kratky camera system. This system includes OptiSAXS confocal maximum beam optics designed specifically for SAXS and a HyPix-3000 Hybrid Photon Counting detector. The sample capillary-to-detector distance was 495.5 mm and was calibrated using silver behenate powder (The Gem Dugout, State College, PA). The useful q-spatial range (4π sinθ / λ, where 2θ is the scattering angle) was approximately q min =0.008Å -1 From q max =0.3Å -1 The X-ray beam energy was 1.2 keV, the Kratky block attenuation was 22%, and the beam diameter was approximately 100 μm.

[0183] Protein samples were loaded into a quartz capillary flow cell using an autosampler, mounted on a stage maintained at 22 °C, and aligned with the X-ray beam. The sample cell and the entire X-ray flight path, including the beam stop, were vacuumed (<1 × 10) to eliminate air scattering. -3 The chamber was kept at a constant temperature (torr). Rigaku SAXSLAB software was programmed for automated data collection of each protein sample and matching buffer, which was rigorously washed with 1 M NaOH before the start of the run and with water and ethanol between samples. Data reduction, including image integration and normalization, and background buffer data subtraction were also performed using SAXSLAB software. After confirming the absence of damage from X-ray irradiation, six 10-minute images were collected from the protein and buffer samples and averaged. An overlay of the SAXS data showed no radiation decay over the 60-minute data collection period. Subtraction of the reference buffer was then performed to obtain the raw SAXS scattering curve from the protein alone.

[0184] Dy-bound Hans-LanM structure determination The anomalous scattering data set was collected at the National Cancer Institute Structural Biology Facility (GM / CA) beamline ID-D at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL). First, the X-ray absorption spectrum was measured (Figure 29), and the 7793.5 (L) peak was selected to maximize the difference in absorption (f). III Two energies were suitably selected: 7760 (edge) and 7760 (pre-edge) eV. Diffraction data at the two energies were collected alternately using an inverted beam geometry with a 30-degree wedge to minimize radiation damage, and then processed in HKL2000.

[0185] The coordinates determined from the high-resolution data were further refined against a new dataset collected at 7793.5 eV using phenix.refine to account for batch variations. In contrast to the high-resolution structure, no heavy elements were found in EF1 of chain D, and the remaining 13 dysprosium ions were retained. No other visible structural changes were observed. Anomaly difference maps were generated in phenix.maps, and the corresponding peak intensities (Table 10) were inspected in Coot. The large difference in the anomalous peak intensities determined at the pre-edge and on-edge energies corresponding to Dy strongly suggests that the metal bound by the EF hand is indeed Dy.

[0186] Maleimide functionalization of agarose beads Maleimide functionalization of amine-functionalized agarose beads has been described previously. Briefly, agarose microbeads (1.2 mL) were dispensed into a 5 mL Eppendorf tube, preconditioned with phosphate-buffered saline (PBS) at pH 7.4, and resuspended in a final volume of approximately 1.7 mL (1.2 mL microbeads and 0.5 mL PBS supernatant). SMCC (0.15 g) powder was dissolved in 3.4 mL of DMSO and combined with the microbeads. After 2.5 h of incubation on a rocker mixer at room temperature, the modified agarose microbeads were washed three times with DMSO to remove unreacted SMCC and three times with coupling buffer (50 mM HEPES, 50 mM KCl, pH 7.0) to remove DMSO. The maleimide microbeads were then used for LanM immobilization within 2 h.

[0187] Breakthrough column experiment An Econo-Column glass chromatography column (Bio-Rad; 5 cm × 0.5 cm) was filled with MilliQ water (18.2 MΩ cm -1), and LanM-microbeads were gravimetrically loaded. The column was washed with 25 mM HCl and MilliQ water and conditioned with Buffer D (10 mM homopiperazine-1,4-bis(2-ethanesulfonic acid), pH 5.0) before breakthrough experiments. RE stock solutions were prepared by dissolving individual RE chloride salts in 1 mM HCl. The stock solutions were diluted with Buffer D. RE solutions were pumped at 0.5 mL / min unless otherwise specified, and column effluent was collected in 1.0 mL aliquots. A wash step with 5 bed volumes of MilliQ water was performed before desorption experiments with the indicated concentrations of chelators or HCl. For single RE ion solutions, RE ion concentrations were quantified using the Arsenazo III assay. Specifically, 40 μL of sample was combined with 40 μL of 12.5 wt% trichloroacetic acid (TCA) and added to 120 μL of 0.1 wt% Arsenazo (filtered) in 6.25 wt% TCA. The absorbance at 652 nm was measured and compared with standards to determine the RE metal ion concentrations. The accuracy of the colorimetric analysis was also confirmed by ICP-MS. For experiments using the RE mixture, the metal ion concentrations were measured by ICP-MS (Table 19, Table 16).

[0188] Table 1. ICP-MS analysis of metallized Hans-LanM samples used for SAXS or crystal structure analysis. Samples for SAXS analysis were incubated with 3 equivalents of metal ion, and the soluble protein was passed through an S75 SEC column to remove aggregates. Sample preparation for crystallography was described herein. The low metal loading of the sample used for crystallography may be related to the additional spin-concentration step and the binding of 3 equivalents to a relatively weak site. Data are presented as means with SD calculated from three technical replicates. JPEG2025532059000028.jpg51169

[0189] Table 2. Hans-LanM La III , Nd III , Dy III , Ca II Summary of fitting parameters for CD titration at Apparent K d (K d,app ), Hill coefficient (n), and change in molar ellipticity at 222 nm (Δ[Θ]) are reported as the mean ± standard error of the fit of data from three independent titrations. Conditions: monomer concentration 15 μM, 25°C, 20 mM acetate, 100 mM KCl, pH 5.0. TIFF2025532059000029.tif35169

[0190] Table 3. RE obtained from analytical SEC (Superdex S75) III -Apparent molecular weight of the Hans-LanM complex Apparent molecular weights were derived from Figure 8 and are also plotted in Figure 2a. Comparison with SEC-MALS data in Table 17. Ionic radii are shown for the 10-coordinate (La) and 9-coordinate (Nd-Ho) complexes described previously. See the legend to Figure 8 for experimental details. TIFF2025532059000030.tif73169

[0191] Table 4. Data collection and refinement statistics for the X-ray structures of La- and Dy-Hans-LanM and Nd-Mex LanM Statistics for the highest resolution shell are shown in parentheses. TIFF2025532059000031.tif235169TIFF2025532059000032.tif71169

[0192] Table 5. SAXS structural parameters of Hans-LanM in the presence of different metal ions The data was obtained using our in-house Rigaku BioSAXS2000 nano The samples were collected at 100°C. Buffer conditions: 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0. Porod volumetric analysis showed that the La and Nd complexes of Hans-LanM were predominantly dimers, while the Dy complexes were only about two-thirds the volume of the La and Nd complexes, supporting the conclusion that they are an equilibrium mixture of monomers and dimers. The R of the Dy and La / Nd complexes was lower than expected based on SEC-MALS. gNote that the difference between the values ​​is small (Table 17 suggests that the hydrodynamic radii of La / Nd vs Dy differ by about 2-3 Å). This is because the protein concentration used for SAXS is 5 times higher than that for SEC-MALS, resulting in a much larger population of Dy-linked dimers in the SAXS experiment. However, these RE-dependent differences are not significant for the SAXS R g The values ​​are within the uncertainty range (see Figure 15). TIFF2025532059000033.tif94169

[0193] Table 6. Analysis using OLIGOMER and CRYSOL software suggests that the Hans-LanM complexes of La and Nd are almost entirely dimeric, whereas the Dy complex is a mixture of monomers and dimers. The OLIGOMER program fits experimental SAXS scattering curves of multicomponent protein mixtures to determine the volume fraction of each component in the mixture. CRYSOL evaluates solution scattering from macromolecules with known atomic structures and fits the experimental scattering curves. Both analyses were performed for two possible states: La-Hans-LanM crystallographic dimers and crystallographic monomers. The analyses showed that the volume fractions of La- and Nd-Hans-LanM complexes are preferentially dimeric, which is likely close to the true distribution since crystallographic dimer models are available. OLIGOMER and CRYSOL analyses under Dy conditions suggest an equilibrium between dimers and monomers. TIFF2025532059000034.tif62169

[0194] Table 7. La of R100K-Hans-LanM III , Nd III , Dy III Summary of fitting parameters for CD titration at Appearance K d (K d,app ) values, Hill coefficients (n), and changes in molar ellipticity at 222 nm (Δ[Θ]) are reported as the mean ± standard error of the fit of data obtained from two (Nd, Dy) or three (La) independent titrations. Conditions: monomer concentration 15 μM, 25°C, 20 mM acetate, 100 mM KCl, pH 5.0. TIFF2025532059000035.tif49169

[0195] Table 8. SEC-MALS analysis of R100K-Hans LanM The concentration of the protein sample loaded onto the column was 3 mg / mL (3.5-fold concentrated compared to the wild-type Hans-LanM sample). TIFF2025532059000036.tif58169

[0196] Table 9. 7760.0 (pre-edge) and 7793.5 eV (L of Dy) III Data collection statistics for the anomalous diffraction data set collected for Dy-Hans-LanM at the edge Statistics for the highest resolution shell are shown in parentheses. TIFF2025532059000037.tif172169

[0197] Table 10. 7760.0 eV (pre-edge) and 7793.5 eV (L of Dy) III The height of the anomalous peak of Dy-Hans-LanM at the edge (unit: e / Å) 3 ) Interestingly, EF2 and EF3 showed the largest anomalous difference map peaks, likely reflecting the biochemical observation that this complex contains only two high-affinity sites (Fig. 1d, Fig. 6). N / A: Not applicable. *No heavy elements are found in EF1 of the D chain of this crystal. TIFF2025532059000038.tif46169

[0198] Table 11. La from Hans-LanM and R100K-Hans LanM III , Nd III , Dy III Concentrations of citrate and malonate required for 50% removal of See Figure 4a and Figure 54. The change in Trp emission intensity of Hans-LanM was monitored at 333 nm. Initial conditions: 20 μM protein, 40 μM RE, 20 mM acetate, 100 mM KCl, pH 5.0, titrated with increasing concentrations of citrate or malonate. Data represent the mean ± standard error (in parentheses) of the fit of data from three independent titrations. TIFF2025532059000039.tif59169

[0199] Table 12. Partition coefficients (D, in bold) and separation factors (SF) of selected REs on immobilized Mex-LanM. The D values ​​represent the distribution of a particular metal ion between LanM and solution in the multielement equilibration experiments described in the text and Materials and Methods ("Batch Experiments to Determine Separation Factors"). Here, a 5 mL feed solution of equimolar REs (La through Dy) (3 mM total RE, 15 μmol total, pH 5.0) is equilibrated with 1 mL of immobilized LanM microbeads (volume: 5.8 μmol for Mex-LanM, 4.1 μmol for Hans-LanM, and 4.7 μmol for R100K-Hans-LanM). Larger D values ​​indicate preferential adsorption to LanM. The separation factor as a function of RE identity is expressed as D. 金属(上) / D 金属(左) The D values ​​were calculated as follows: An SF of 1.0 indicates no selectivity for intra-RE separation, while an SF much greater (or less) than 1 indicates a preference for one ion over another. See Figure 4c for a plot of the base-10 logarithm of the D values ​​for each metal ion. Uncertainties are shown in parentheses, with the values ​​in parentheses representing the uncertainty to the nearest significant figure: for example, 0.86 (1) means 0.86 ± 0.01, and 2.15 (32) means 2.15 ± 0.32. For HREs such as Dy, especially Hans-LanM and R100K-Hans-LanM, the uncertainty is larger because the amount of RE adsorbed on the column is much smaller than that of LREs due to weak binding to LanM. The uncertainty in the D values ​​is the standard deviation from three independent column runs. The uncertainty in the SF values ​​was calculated by error propagation of the corresponding D values. JPEG2025532059000040.jpg110169

[0200] Table 13. Partition coefficients (D) and separation factors (SF) of selected REs on immobilized Hans-LanM. See the legend in Table 12 for details. JPEG2025532059000041.jpg93169

[0201] Table 14. Partition coefficients (D) and separation factors (SF) of selected REs on immobilized R100K-Hans-LanM. See the legend in Table 12 for details. JPEG2025532059000042.jpg92169

[0202] Table 15. Amino acid and DNA sequences of constructs used in this study The first residue (after cleavage of the N-terminal Met) of the cytoplasmically expressed Hans-LanM protein is A24, the predicted signal peptide cleavage site according to SignalP 6.0. All residues are numbered based on the full-length sequence. TIFF2025532059000043.tif242169

[0203] Table 16. Metal ion concentrations in synthetic feed solutions for on-column Nd / Dy separations in Figures 4d and 55 The concentrations were determined by ICP-MS analysis. TIFF2025532059000044.tif20169 Example 2

[0204] This example describes the use of the peptides / proteins of the present disclosure.

[0205] The structures of Hans-LanM and Mex-LanM provide insight into the mechanism of Hans-LanM dimerization, as well as insight into the mechanisms for enhancing metal binding selectivity, stoichiometry, and cooperativity, as well as the mechanism for the K dimer This suggests several routes to globally modulating the dimerization equilibrium away from intracellular systems and toward the high-concentration regime suitable for industrial processes.

[0206] The increase in selectivity exploits the additional stabilization of the metal-protein complex provided by dimerization by identifying the point in the lanthanide series where there is a significant change in dimerization propensity (i.e., the point at which a complex with one lanthanide(III) ion (e.g., Tb) stabilizes the dimer substantially more than the next lanthanide ion (e.g., Dy). Because the position of the dimerization equilibrium depends on the monomer concentration, K dimer Being able to adjust the Λ is important to reflect the protein concentration in the intended conditions of use (mostly to weaken dimerization affinity).

[0207] Higher stoichiometry allows for more economical REE binding (better atom economy), while higher cooperativity may lead to sharper desorption profiles and, consequently, better separations.

[0208] Improved resolution using Hans-LanM The response of Hans-LanM to Sm(III), Gd(III), Tb(III), and Ho(III) was determined using CD spectroscopy and plotted in Figure 1e and Table 2 (Figure 37, Table 20). The experimental conditions were the same as described above (15 μM Hans-LanM, 30 mM acetate, 100 mM KCl, 10 mM EGTA, 0-10 mM Ln(III), pH 5.0).

[0209] Table 20. Extension of Table 2 (including Sm, Gd, Tb, and Ho) Values ​​reported with uncertainties were performed at least twice. TIFF2025532059000045.tif46169

[0210] These data follow the general trend described above in which the magnitude (Δ[Θ]) of the protein response to Dy(III) is smaller than that to La(III) and Nd(III), and this decrease in magnitude occurs approximately with Sm(III). This is due to the apparent K d This also correlates with the rapid decrease (2.5-3 times) in apparent K between Tb(III) and Dy(III). dA second sharp decrease (5-fold) in the EF3 binding ratio occurred, which would be an industrially important separation. It is currently unclear whether the Hill coefficient of approximately 2 for La(III)- and Nd(III)-bound Hans-LanM (Table 2) reflects metal binding to EF3 and dimerization of two monomers or EF2 / 3 from a single monomer. One interpretation of the low cooperativity of Dy(III) (and other MREE-HREE) binding is that it is indeed metal-dependent dimerization that leads to cooperativity. These results motivate us to explore the use of dimerization equilibria in variants of this protein to perform this separation.

[0211] To do this, it is important to understand the interactions that contribute to the stabilization of the dimer. The crystal structure of Hans-LanM suggests that the hydrogen bond network centered around R100 plays a key role, but other interactions at the interface may also play a role (e.g., M92, I43, I47, L96). This suggestion is supported by ITC analysis of the R100K variant (Table 21). While the presence of glycerol in these experiments may have some effect on the quantitative thermodynamic values, the data suggest that at R100K, the K of the La(III) complex is dimer showed a significant increase from <0.4 μM to 14 μM, while the K dimer The values ​​are similar to those of the wild-type protein, further demonstrating that R100-dependent dimerization selectively enhances the stability of the La-Nd complex.

[0212] Table 21. Thermodynamic parameters of WT and R100K Hans-LanM obtained by fitting ITC thermograms to the dimer dissociation model. These experiments were performed similarly to those shown in Table 18, except that the buffer contained 5% glycerol. TIFF2025532059000046.tif34169

[0213] Therefore, for the dimerization equilibrium to operate at millimolar protein concentrations (desirable for separation), some of the other interactions that contribute to dimerization must be eliminated. To achieve this, some of the following variants (individually or possibly in combination) are pursued: M92L, M92A, or M92D: LanM_012 contains a leucine at this position, and significant dimerization is not observed in the absence of an REE (see below). These mutations may reduce the hydrophobicity of the central dimer interface and thus reduce the residual Hans-LanM dimerization identified in ITC and other studies. A44N, A44S, or A44T: These mutations are hypothesized to add a hydrogen bond between this residue in one monomer and, for example, the T7 residue of EF2 or EF3 in an adjacent monomer. If the interaction occurs with EF2, this would allow specific interactions with both EF2 and EF3 in the dimer, linking dimerization to the occupancy of both EF2 and EF3, possibly increasing the cooperativity and affinity drop-off (observed in Figure 37). D93N, D93A, or D93E: The D93N or D93A mutation likely weakens the interaction of R100 with the adjacent monomer, such that only / predominantly hydrogen bonds to the metal ligand contribute to dimerization in this region of the protein. D93E may alter the selectivity of REE-induced dimerization by changing the hydrogen-bonding network and altering the REE at which dimerization is most robust.

[0214] To be able to immobilize a Hans-LanM version that can dimerize on a column, the two monomers can be linked using a linker (such as a polypeptide sequence) of appropriate length and flexibility. Examples of possible constructs are shown below (the first one is likely too short to support intramolecular dimerization; the linker sequence is underlined, the rest of the sequence is that of Hans-LanM): TIFF2025532059000047.tif169169

[0215] The dimerization and linker variants can be combined for separation experiments on a column or in solution at higher protein concentrations.

[0216] Metal binding stoichiometry of M. extorquens LanM Previous studies on M. extorquens LanM (Featherston et al. JACS 2021; Mattocks et al. Chem. Sci. 2022; Dong et al., ACS Cent. Sci. 2021) have shown that the third binding equivalent assigned to EF1 is kinetically less stable than the first two, which manifests as a binding stoichiometry of 2 under low pH and on-column conditions. For example, at pH 5, Nd 3+ Apparent K of the structural changes of EF-hands 2 and 3 against d is 21 pM, while that of EF1 is 4.1 nM (Mattocks et al., Chem. Sci. 2022). Therefore, we sought an approach to make the affinity of EF1 comparable to that of EF2 / 3, thereby increasing the stoichiometry to (at least) 3. A common approach is to install unique hydrogen bonds between the EF hands in the folded state of the protein, thereby increasing affinity.

[0217] The crystal structure of Nd-Mex-LanM shows a hydrogen bond between D56 (the "-3" residue of EF2; the minus sign indicates that it is located before the loop) and K93 (the 10th residue of EF3). However, no equivalent interaction exists for the EF1 / 4 pair (A32 at the -3 position of EF1 and A117, the 10th residue of EF4). We hypothesized that introducing an equivalent hydrogen bond into this pair might contribute to stabilizing EF1. We made substitutions A117K and A117R in the A32D background, and these two proteins (A32D / A117K and A32D / A177R) were expressed and purified from E. coli in the same manner as wild-type Mex-LanM. 3+The protein's response to was characterized by CD at pH 5.0 (Figures 39 and 40) for direct comparison with wild-type data (Mattocks et al., Chem. Sci. 2022).

[0218] Both permutation sets have apparent K d Although the A32D / A117R variant appears to behave better, the overall response (Figure 4C, right) is slightly asymmetric in A32D / A117R, suggesting that EF1 exhibits an improved response relative to the wild-type (see Figure 2), but is not as similar as EF2 / 3 in A32D / A117K. In A32D / A117K, the Hill coefficient values ​​suggest that EF1 likely does not respond cooperatively with EF2 / 3. It is not yet clear whether the improved response is sufficient to increase the binding stoichiometry or whether this site is still kinetically less stable than EF2 / 3. Initial xylenol orange competition experiments suggested that A32D / A117R destabilizes metal binding relative to its overall equivalent, while A32D / A117K is similar to WT and I42L / N108D / I115L is somewhat stabilizing relative to WT (Figure 38, bottom). Reproduction of these results suggests that A32D / A117R and A32D / A117K perform similarly and somewhat better than WT (see below and Figure 68). The binding stoichiometry will be tested at different pHs using spin columns to separate bound and unbound metal ions, and also using Cys-modified versions immobilized on agarose beads, as described previously. The best variant combinations (e.g., A32D / A117K and I42L / N108D / I115L) will be tested as well.

[0219] In the crystal structure with almost the same EF loop structure (except for the different hydrogen bonding pattern induced by the N1 residue), EF4 is 3+The observed occupancy of EF3 / EF4 suggests that the low stability of this site may be due to the lack of structural changes induced by metal binding. The structure reveals that the helix between EF3 and EF4 is slightly unwound, beginning at A98 (which may be due to EF4 occupancy). Adding a helical break in this region (i.e., replacing A98, A99, V100, or possibly A102 with Gly) could optimize the structure of EF4 and separate it from EF3, resulting in a conformational change sufficient to increase affinity. Similarly, the helix between EF1 and EF2 has a small break at G51, which may explain why EF1 behaves almost independently of EF2. The G51A substitution may link EF1 to EF2 / 3, increasing the Hill coefficient to 3. The fluorescence of Y96 at around pH 5 is a way to examine the effect of these mutations. This decreases with the addition of the first two binding equivalents, but increases with the addition of the third, as if the third binding equivalent slightly destabilizes the folded structure of the protein. Mutations that retain low Tyr fluorescence even with the addition of the third equivalent are sought.

[0220] The combination of these mutations on these two helices, along with the A32D / A117K and / or I42L / N108D / I115L variants mentioned above, allows for a tight-binding stoichiometry of up to 4, as well as the connection of all four EF-hands into a single cooperative unit (n up to 4), which could significantly improve the separation of adjacent REs. If this were not possible, activation of EF4 and its cooperative binding with EF1 could yield two halves of the protein, each with n = 2, which would still significantly improve protein performance.

[0221] Conceptually related to the hydrogen bond linking EF1 and EF4 is the use of a disulfide. As shown in Figure 27 above, natural LanMs possess this property, and the metal-binding properties of these proteins are explored. An example of such a protein has the following sequence (after cleavage of the putative signal sequence; the EF hands are underlined): TIFF2025532059000048.tif18169

[0222] LanM_002 is Sm than LanM_001. III ,EU III , Tb III , Dy III It is a strong sensitizer for luminescence EU III Based on the luminescence lifetime experiments and confirmed by the X-ray structure, the observation that Hans-LanM (LanM_002) has zero coordinated water molecules and the presence of two Trp residues near EF2 / 3 suggests that Eu III Not only that, but Sm III and Dy III These results suggest that Hans-LanM may be able to sensitize the emission of ions with short emission lifetimes, such as Eu(III) and Tb(III) (Figures 41-43). As shown in Figures 41 and 42, Hans-LanM can sensitize Eu(III) and Tb(III) under time-resolved and steady-state conditions, where Mex-LanM performs much worse. Figure 43 shows that the emission of Sm(III) and Dy(III) bound to Mex-LanM is extremely poor, whereas Hans-LanM produces detectable signals. This may enable sensitive detection of these metals even in complex environments (similar to Tb detection using Trp-LanM) and within cells.

[0223] Characterization of Xanthobacter flavus LanM (LanM_012) The LanM sequence alignment combined with the structure allowed us to explore LanM proteins that may exhibit additional hydrogen-bonding interactions between other EF hands. This may amplify the protein's susceptibility to dimerization (between lanthanides), alter dimerization strength, and allow for increased cooperativity (as more EF hands are interconnected). In the crystal structure, EF3 of one monomer interacts with EF3 (and EF4, but it is Na) of the other monomer. + The interaction appears to be primarily linked to the occupancy of the nucleotide sequence of LanM_012 (occupied by EF1). H25 (three residues after EF1) and T40 (the sixth residue of EF2) interact via a hydrophobic interaction between the imidazole ring of His and the methyl group of Thr. Converting this interaction to a hydrogen bond may further stabilize the interaction. In the X. flavus sequence, H25 is Asp and T40 is Lys, suggesting that they may be hydrogen-bonded. The moiety corresponding to T29 is Lys, which may also be involved in hydrogen bonding. The peptide sequence of LanM_012 for cytoplasmic expression (with the signal peptide removed and the residues corresponding to H25 and T40 of Hans-LanM underlined) is as follows: TIFF2025532059000049.tif17169

[0224] Gene sequence of LanM_012 inserted into pET29a, similar to Hans-LanM. TIFF2025532059000050.tif37169

[0225] LanM_012 was expressed and purified in the same manner as Mex-LanM, and approximately 10 mg / L was obtained. Adding Ala as the first residue after the N-terminal Met may improve expression according to the N-terminal rule. As shown in Figure 44, the apoprotein elutes from the SEC column with a retention volume consistent with the monomer, while the La and Dy complexes elute as apparent dimers. The tendency of this protein to dimerize indicates that the presence of residues corresponding to R100 is highly likely to predict the dimerization ability of LanM, but other interactions that are not yet clear are involved in the overall strength of dimerization and the difference in the dimerization tendency between apo- and REE-bound proteins.

[0226] Similar to Hans-LanM, the third binding equivalent of LanM_012 is relatively weak and cannot exceed xylenol orange (which has an approximately micromolar affinity for RE) (Figure 45). However, apo-LanM_012 was found to have helical properties comparable to the LRE-binding state of Hans-LanM and Mex-LanM at room temperature (Figure 46).

[0227] The apoprotein has an important secondary structure at room temperature, but temperature-dependent CD experiments show that the stability of the helix strongly depends on temperature and the presence of metal, with the denaturation temperature increasing in the order of apo < Dy < La, indicating an improvement in the stability of the LREE complex (Figure 47). This suggests that it may be possible to utilize high temperature to selectively desorb HREE from the protein.

[0228] Titration of the protein with buffered metal solutions and competitive titration with citrate (Figure 48, left and right) indicated that LanM_012 binds REEs more strongly than Hans-LanM. Interestingly, as shown in the fitting parameters associated with these data (Tables 22 and 23), binding of both La(III) and Dy(III) is cooperative (n ≈2), likely a result of complete dimerization in the presence of both ions (Figure 44; compared to Hans-LanM, which is primarily monomeric under these experimental conditions). However, it is noteworthy that the ΔF values ​​for Nd and Dy are identical and distinct from those for La. This suggests conformational differences between the La- and Nd-bound proteins, which may be exploited for the separation of REEs (Table 23).

[0229] Table 22. Fitting parameters for Figure 48 (left) TIFF2025532059000051.tif29169

[0230] Table 23. Fitting parameters for Figure 48 (right) TIFF2025532059000052.tif39169

[0231] Finally, the similarity between the EF-hand sequences of LanM_012 and Hans-LanM suggested that LanM_012 might also lack coordinating solvent, resulting in REE complexes with not only enhanced luminescence but also high affinity, desirable for sensing applications. This prediction is verified by the results shown in Figure 49. Furthermore, these data predict that the presence of Glu at position 9 of the EF-hand (possibly in combination with Asn at position 1) could result in REE complexes with no coordinating solvent.

[0232] Sequences (additional sequences are shown in Table 15) TIFF2025532059000053.tif130169

[0233] Figure 27 contains a non-exhaustive list of sequences that may dimerize (including residues 72, 78, and 80 shown in the figure (which correspond to D85, E91, and D93 in the figure in the main text), as well as the residue corresponding to R100 (position 87 in the figure). These sequences are compared with sequences that are unlikely to dimerize, such as M. extorquens, and several other specific sequences described in the legend. These include: TIFF2025532059000054.tif72169

[0234] A non-limiting set of variants is as follows: TIFF2025532059000055.tif170169

[0235] Key features for RE-dependent protein dimerization Based on the results, it is expected that the following properties are sufficient for dimerization by the described mechanism: 1) LanM-like sequences. First, although most have four EF-hand motifs, proteins with two or three EF-hands are possible, with the second and third properties described below (however, such proteins may or may not dimerize via distinct mechanisms / interfaces). Second, except for the case of three EF-hand proteins (in which one of the EF-hands is disrupted so that carboxylate residues are almost absent), the EF-hands are separated by approximately 12–13 residues (12–13 residues between the terminal amino acid of one EF-hand, usually Glu, and the first amino acid of the next EF-hand, usually Asp or Asn). This spacing is, to our knowledge, unique among EF-hand proteins and may itself be diagnostic of LanM. Third, at least one EF-hand contains a proline in the second position (in Mex-LanM, all four EF-hands have a P2 residue). Using the first two criteria and a sequence length of <200 residues, we identified 696 putative LanMs. 2) The following residue pattern at sequence positions aligns with D85, E91, D93, and R100 of Hans-LanM (where D85, E91, and D93 correspond to positions 3, 9, and 11 of EF-hand 3, and D93 and R100K appear to be unique residues that enable sensitive dimerization upon identification of a protein-bound RE). While the structure indicates that other interactions are also involved in stabilizing the dimer, the above residues are conserved in many LanM proteins and appear to be key for RE-sensitive dimerization.

[0236] Note that, in principle, the entire protein sequence is not necessarily required for RE-dependent dimerization; for example, not all residues within or near EF1 and EF4 of the primary sequence are required. In other words, it is envisioned that shorter (more atom-economical) LanM-like proteins may also be capable of RE-sensitive dimerization.

[0237] Furthermore, the trend in inter-REE selectivity, the steepness of inter-REE selectivity, and the overall strength of dimerization can be modulated (decreased or increased) by mutagenesis of polar and non-polar residues at the interface, as described herein.

[0238] Finally, other conceptually related mechanisms of dimerization may be possible in other lanmodulins (e.g., use of a different dimerization interface, exploiting specific interactions between EF2 ligands) and indeed in other lanthanide-binding proteins. Example 3

[0239] This example describes the use of the peptides / proteins of the present disclosure.

[0240] LanM_013 is interesting because EF2 resembles the Hans-LanM EF hand (N1 and E9 residues, with Gly at position 4), whereas EF3 resembles the Mex-LanM EF hand, with all EF hands containing a Pro residue. It was hypothesized that the structure of EF2 might be highly sensitive to the identity of the RE (as is the case with the Hans-LanM EF hand), but the protein is not expected to dimerize, potentially enhancing selectivity among the lanthanide series within a single LanM unit. TIFF2025532059000056.tif54169

[0241] The procedure used for expression in E. coli and purification of LanM_013 was identical to that used for Mex-LanM, except that Ca(II) was not included in the gel filtration step. Figure 56 shows the high purity of the protein after this purification procedure. The elution volume of the protein from the S75 column was consistent with that of the monomer. Purification yielded 80 mg of LanM_013 from 2 L of culture (40 mg / L). The protein contains two Tyr residues and has an extinction coefficient at 280 nm of 2980 M -1 cm -1 was used.

[0242] Figures 57-59 relate to the binding stoichiometry of LanM_013. In Figure 57, for clarity, peak absorbance is tracked at both 278 nm and 285 nm. Both of these peaks reach a maximum after the addition of 2.0 equivalents of La(III). Note that this assay specifically reports on the environment of tyrosine residues in the protein in the vicinity of EF-hands 2 and 3. The total amount of metal equivalents bound to the protein may be higher.

[0243] Figure 58 shows the fluorescence emission of the Tyr residue of LanM_013 upon titration with La(III), Nd(III), and Dy(III). Similar to Mex-LanM, an initial decrease in fluorescence occurs with the addition of the first 2 equivalents of La(III) and Nd(III), followed by an increase at approximately 1.0 equivalent, indicating that under these conditions, the protein can bind at least 3 equivalents of La(III) and Nd(III). Interestingly, this is not observed with dysprosium, indicating structural differences near one or both of the protein's Tyr residues when LanM_013 binds specific lanthanides. Figure 59 uses competitive titration of La(III)-bound LanM_013 (3 equivalents of La(III)) with citrate, monitored by intrinsic tyrosine fluorescence, to estimate how tightly La(III) binds to the protein at its three highest affinity sites. The initial loss of fluorescence is due to the weaker binding of the third metal equivalent (see Figure 57). Even 100 mM citrate was unable to restore tyrosine fluorescence to that of the apoprotein. This indicates that the remaining two metal equivalents presumably bound to EF2 and EF3 are tightly bound (perhaps even tighter than in Mex-LanM). For comparison, the tightest two equivalents of La(III) desorb by about 50% at about 20 mM citrate (see Figure 4a in the Hans-LanM study). Slower equilibration kinetics may also play a role in the LanM_013 system.

[0244] Figures 60 and 61 relate to the stoichiometry and affinity of LanM_013. In Figure 60, xylenol orange was used as a colorimetric competitor for La(III) and Dy(III) binding. The results show a slight increase in signal throughout the Dy(III) titration, with a significant increase occurring between 1 and 2 equivalents of added metal, indicating relatively weak binding to Dy(III) compared to La(III). La(III) clearly exhibits approximately 2.0 equivalents of binding, which corresponds well with the other experiments described above. The binding of the third equivalent of La(III) revealed by the fluorescence experiments is likely weak: if it were weaker than approximately 1-10 μM, it would not be observed in the xylenol orange assay. In Figure 61, CD spectroscopy is used to assess the extent of conformational changes in LanM_013 upon lanthanide binding. A clear titration endpoint was observed for both La(III) conditions (pH 5 and pH 7), whereas a more extensive response was observed for Dy(III). This suggests that the affinity of at least one site in the Dy(III)-LanM_013 complex is likely weaker than that of the La(III)-LanM_013 complex. In Figure 62, CD spectroscopy was used to determine the apparent K for the response of LanM_013 to certain lanthanides. d The values ​​are preliminarily estimated to be 11 pM (La) and 96 pM (Dy), with Hill coefficients of approximately 1.5 for both.

[0245] These experiments allow us to draw conclusions even in the absence of more detailed affinity measurements. LanM_013 possesses several intriguing properties that may be suitable for LRE-only recovery or LRE / HRE separation. Metal binding to the two tightest binding sites (presumed to be EF2 / 3 based on other characterized LanMs) appears to result in a sufficiently distinct conformation near the immediately succeeding Tyr (Y73) that the fluorescence emission of that residue is quenched in the presence of La(III) and Nd(III) but not Dy(III). Other biophysical data also suggest a larger affinity difference between the heavy and light REs at at least one site, but the location of the affinity "breakpoint" in the lanthanide series needs to be assessed. In this respect, this protein may have achieved a Hans-LanM-like effect within a single polypeptide. The two tightest sites appear to allow tighter LRE binding than Mex-LanM.

[0246] The insights gained from the crystallographic analysis of the Mex-LanM and Hans-LanM systems allowed us to make predictions regarding the origin of key properties of this novel protein and, therefore, potentially extend it to proteins with similar sequence characteristics that have yet to be biochemically characterized. First, regarding the apparent structural differences between Dy-bound LanM_013 and La / Nd-bound LanM_013: the E9 residue (E44) of EF2 may have changed from bidentate (with the LRE) to monodentate (with the HRE), which may somehow be transmitted to EF3, and EF2-EF3 pairing may only occur when the LRE is in each EF hand. The presence of a second Gly (G39) in EF2 may also play a role in generating a conformation of the HRE bound to EF2 that is incompatible with the interaction with EF3. The first residue of EF2, Asn, may also contribute. Second, based on the citrate data, the affinity of this protein for La(III) is clearly higher than that of Mex-LanM, suggesting that a potential cause of the higher affinity may be additional hydrogen-bonding interactions specific to the LRE-bound form of the protein. Based on comparison with the Mex-LanM structure, residue E74 (Glu immediately following EF3) may be well-positioned to hydrogen bond with K29 and / or K25. Such interactions may also contribute to the difference in affinity of the protein for LRE versus HRE complexes. Example 4

[0247] This example describes the use of the peptides / proteins of the present disclosure.

[0248] Nd and Yb sensing using LanM_012 Mex-LanM(T90W), Hans-LanM(R100K), and LanM_012 were tested for their ability to enhance the near-infrared (NIR) emission of Nd(III) and Yb(III). The data are shown in Figure 63. The data indicate that all three sensors can enhance Nd(III) and Yb(III) at pH 4.0 and above. Only LanM_012 showed appreciable intensity for Nd(III) at pH 3.0, while none of the sensors were able to enhance Yb(III) at pH 3.0. This may be due to the low binding affinity of LanM to the later REE at low pH. Overall, LanM_012 exhibits the best performance for both ions, but exhibits particularly high emission intensity for Yb(III). Therefore, all the findings contained in this paper suggest that LanM_012 is a particularly promising protein-based sensor for several individual REEs (Tb, Eu, Dy, Sm, Nd, and Yb), which may be useful for in vitro REE studies and for REE uptake studies in cells.

[0249] Dissociation constant (K d s) determination The apparent dissociation constants of LanM_013 for three middle- to late-REEs with similar ionic radii were determined at pH 5.0 (summarized in Table 24 and plotted in Figure 64). LanM_013 prefers light lanthanides such as Gd(III) over heavier lanthanides such as Dy(III) and Ho(III). These titrations with a single REE show fairly steep affinity trends over a narrow range of atomic numbers: there is an approximately 7-fold difference between Gd and Ho (only Tb and Dy lie in between). The apparent K of LanM_013 for Gd is dis about three times tighter than Dy, which is about 2.5 times tighter than Ho. Furthermore, there appears to be a tendency for lighter lanthanides to be more cooperatively bound than heavier lanthanides. LanM_013 exhibits a biphasic response to Gd(III), with a cooperative major phase and a non-cooperative minor phase (Figure 64A). Similar curves may be observed for Dy(III) and Ho(III) if measured at higher metal concentrations, since this potential second phase is not considered in the fitting for Dy and Ho, thus limiting the apparent K d s may differ slightly from the values ​​reported here. As in other LanMs that have been studied, it is currently unclear whether the weak phase is EF1. d It should be noted that s is slightly tighter than previously reported, possibly because a higher protein concentration (20 μM vs. 10 μM) was used previously, which may have had a slight effect on the free metal concentration in the titration, resulting in a slight overestimation of the calculated free metal concentration.

[0250] Table 24.Gd III , Dy III , Ho III Summary of fit parameters for CD titration of LanM_013 by TIFF2025532059000057.tif66169

[0251] On-column intra-REE selectivity of LanM_013 compared with other orthologs To quantitatively measure the inter-REE selectivity of different lanmodulins, equilibrium binding was performed with the immobilized protein using a light REE mixture (La-Dy, Figure 65) or a heavy REE mixture (Gd-Lu[containing Y], Figure 66) at equimolar concentrations (pH 5). Each solution was circulated on the LanM column for 2 h. The distribution of REEs between the solid phase (i.e., the LanM-bonded phase) and the aqueous phase was quantified to determine the distribution value (D). The D value was used to determine the separation factor by taking the ratio of the distribution values ​​between the two REEs of interest. LanM_001 exhibited the highest selectivity for the LREEs and MREEs Ce, Pr, Nd, Sm, and Eu, forming a selectivity plateau with the smallest difference between the D values. LanM_002 and LanM_012 showed a narrow selectivity plateau centered around Pr, significantly improving the selectivity for LREEs and HREEs. LanM_013 showed intermediate selectivity trends compared to LanM_001 and LanM_002 / LanM_012, including preferential binding to Ce-Sm. Interestingly, among the variants, LanM_013 exhibited the steepest selectivity for Eu to Dy, with separation factors close to 2 for Eu / Gd, Gd / Tb, and Tb / Dy. Compared to LanM_001, these variants are able to more effectively separate adjacent REEs from Pr to Dy. Notably, the Gd-Lu,Y results for LanM_013 (Figure 66) appear to show selectivity trends much closer to those of LanM_001 than would be expected from the La-Dy data (Figure 65). The presence of LREEs may enhance selectivity between HREEs, possibly through a mixed binding scheme.

[0252] Experimental details: Batch experiments to determine partition values ​​and separation factors The LanM-immobilized microbeads were washed with DI water. 5 mL of feed solution (3 mM total REE, equimolar (pH 5)) was added to 1 mL of microbeads and incubated for 2 h. The equilibrated liquid was collected and the REE concentration was calculated as [M]. adsorption Next, the REEs were desorbed using 4 mL of 0.1 M HCl, and their concentrations were measured as [M]. desorptionThe REE distribution (D) between the LanM phase and the solution phase was calculated as follows: JPEG2025532059000058.jpg17155, where [M] LanM and [M] Liquid are the metal ion concentrations in the LanM solid phase and unbound solution phase at equilibrium, respectively. To account for free liquid absorbed within the agarose microbeads, the following correction was made: JPEG2025532059000059.jpg10143The separation factor (SF) is defined as: JPEG2025532059000060.jpg16153where, D REE1 and D REE2 are the distribution values ​​of REE1 and REE2, respectively.

[0253] Improves the binding ability of Mex-LanM by stabilizing metal binding to EF1 and EF4 The target sites that enhance the binding stoichiometry are EF1 and EF4. EF4 exhibits very weak metal binding, whereas the affinity of EF1 is slightly lower than that of EF2 and EF3 at neutral pH, but decreases significantly as the pH decreases. The increased instability of EF1 is hypothesized to be due in part to its pairing with EF4, which does not bind metal ions as tightly and is therefore more pH-sensitive. Furthermore, the lower affinity of EF4 may be related to preorganization (i.e., the adjacent helices are stabilized by metal binding to EF2 and EF3).

[0254] To achieve stronger REE binding to EF1 and EF4, we employed three different site-directed mutagenesis strategies. First, we optimized the metal-binding loops of EF1 and EF4 by replicating the amino acid sequence found in EF2 and EF3. In particular, EF4 has an Asn occupying position 1 instead of Asp1 found in the other three EF-hands (hereafter, subscripts indicate positions within the EF-hand). The non-binding side chain NH2 of Asn1 cannot form a hydrogen bond with the backbone NH of Gly6, leading to a different conformation of the loop. Perhaps for this reason, position 8 in EF1 and EF4 is Ile, rather than Leu8 as in EF2 and EF3. Therefore, mutations that change Asn1 in EF4 to Asp and Ile8 in EF1 and EF4 to Leu may optimize the binding geometry of lanthanides (variant group 1: N108D and I42L N108D I115L).

[0255] Furthermore, introducing a hydrogen bond between EF1 and EF4 could enhance the stability of the complex in a metal-dependent manner. In particular, the crystal structure of Nd(III)-bound Mex-LanM revealed a hydrogen bond connecting position -3 (D56) of EF2 and position 10 (K93) of EF3. Meanwhile, Ala residues occupy equivalent positions in EF1 and EF4. Therefore, mutating these Ala residues in a manner similar to EF2 and EF3 could potentially establish a stabilizing hydrogen bond (variant group 2: A32D / A117K and A32D / A117R).

[0256] EF4 is presumed to be preorganized in such a way that less energy is released during metal-induced conformational changes. Therefore, we hypothesized that destabilizing the apo-binding loop would improve the stoichiometry. This would activate EF4 for metal binding, thereby stabilizing the metal-bound conformation of EF1. In wt-Mex-LanM, occupancy of EF2 and EF3 contributes to the majority of the protein's α-helical content. It has been proposed that the α-helix (α2) between EF3 and EF4 is formed entirely or largely upon REE binding to EF2 / 3; therefore, preventing the complete formation of α2 during metal chelation between EF2 and EF3 may result in some stabilization when EF4 is occupied. Therefore, three Ala residues near the center of α2, which tend to stabilize helices, were selected for mutation to more flexible Gly residues, which tend to destabilize helices (variant group 3: A98G, A99G, A102G).

[0257] The binding stoichiometry of the variants was investigated using a xylenol orange (XO) competition assay and circular dichroism (CD) spectroscopy. The buffer used for the XO competition assay was 20 mM acetate, 20 mM MES, 100 mM KCl, pH 6.1. CD spectroscopy was used to observe the conformational changes of the variants upon Nd titration. The buffer used for CD was 20 mM acetate, 100 mM KCl, pH 5.0.

[0258] Variant Group 1None of the variants showed improved binding stoichiometry. However, apo N108D was found to have significant α-helical content (almost half of the maximum helicity) at pH 5.0 (Figure 67B). If Asp were present at this position in EF4, it could form a hydrogen bond with the backbone NH of Gly at position 6, generating an α-helix around EF4. Conceivably, these generated α-helices could be located between EF3 and EF4 and at the C-terminus after EF4. The I42L and I115L mutations, along with N108D, appear to further stabilize the apo structure, but the effect is small.

[0259] Variant Group 2 The A32D / A117R and A32D / A117K variants showed an improvement in binding stoichiometry of approximately 0.5 equivalents at pH 6.1, as determined by XO competition assays (Figure 68A). This same effect was not observed in CD titrations, but it should be noted that this may reflect the pH dependence of hydrogen bond strength, as CD titrations were performed at pH 5.0 (Figure 68B). The secondary structure of these variants is similar to that of wt LanM. The additional hydrogen bonds may contribute to stabilization of the EF1 and EF4 complex, but further mutations (e.g., stabilization of metal-bound EF4) may be required to achieve the full effect.

[0260] Variant Group 3 None of the variants showed improved binding stoichiometry. A98G and A99G appear to destabilize EF1 binding (and possibly EF2 / 3), while A102G has minimal effect on stoichiometry (Figure 69A). However, all A to G variants exhibit both reduced α-helicity in their apo state and less helicity than the wt in the metal-bound state (Figure 69B). The reduced helicity in the apo state in these variants suggests that α2 is partially ordered in the wt apoprotein or serves to stabilize another portion of the protein (e.g., the α-helix after EF4 to the C-terminus).

[0261] "Combo" Variant Group We prepared a variant group called "combo" by combining mutations from variant groups 1 and 3 (Figure 70). Among the alternatives, A99G was selected because the CD titration curves suggested an improved secondary binding event (Figure 71B). Interestingly, the combination of A99G and N108D mutations reduced the α-helicity of apo N108D to near the wt level. This observation suggests that directly or indirectly endowing residue 108 with the ability to hydrogen bond with the G113 backbone NH, leading to communication and stabilization of the α2 helix and also to communication with the C-terminal helix. On the other hand, the variant containing A99G may not be an appropriate mutation location because it did not show a complete conformational change or improved binding stoichiometry. The variant I42L A99G N108D I115L showed a slight improvement in Nd binding stoichiometry in the XO competition assay, but CD results showed a decrease in binding stoichiometry at pH 5.0.

[0262] Therefore, to stabilize metal binding to EF1 and EF4, the results suggested the following. First, A32D, which has a basic residue (presumably R) at residue 117, slightly stabilizes EF1, but not enough to result in stabilization below pH 5. Further stabilizing EF4 metal binding, it is conceivable that the A32D / A117R mutations could be combined with EF4-stabilizing mutations to also fully stabilize EF1. Second, N108D is essential for forming an optimized EF4-binding loop, but it appears necessary to combine it with a mutation that destabilizes the helix within the apoprotein. Strategically introducing this destabilizing Gly residue seems important, but A99G may not be the right choice. We left the N108D mutation intact and introduced an A-to-G mutation in the region following EF4. Two Ala residues following Pro123 were chosen: variants N108D A124G and N108D A127G, which can be expressed, purified, and subsequently characterized in a similar manner. The variant N108D A102G can also be investigated. Finally, because Hans-LanM and LanM_012 feature EF hands with an Asn at position 1 (although the position of the Gly residue within the loop is different), partial loop sequences from these proteins may also be useful for substitution into EF4. TIFF2025532059000061.tif63169

[0263] Although the present disclosure has been described with reference to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

1. A protein capable of binding metals and / or metal ions, comprising a first EF hand motif, a second EF hand motif, a third EF hand motif, and a fourth EF hand motif, each EF hand motif comprising 11, 12, or 14 amino acid residues; wherein the first EF hand motif, the second EF hand motif, the third EF hand motif, and the fourth EF hand motif have 12 amino acid residues, each EF hand motif has the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E where i) First EF hand motif, second EF hand motif, and fourth EF hand motif each X 1 is independently D or N; each X 2 are independently any canonical amino acid; each X 3 is independently D, N, or E; each X 4 are independently any canonical amino acid; each X 5 is independently D, N, or E; each X 6 are independently any canonical amino acid; each X 7 are independently any canonical amino acid; each X 8 are independently hydrophobic residues; each X 9 is independently D, E, or T; each X 10 are independently hydrophobic residues; and each X 11 are independently any canonical amino acid; ii) Regarding the third EF hand motif X 1 is N; X 2 is any canonical amino acid; X 3 is D; X 4 is G or A; X 5 is D or N; X 6 is any canonical amino acid; X 7 is T or S; X 8 is a hydrophobic residue; X 9 is E; X 10 is a hydrophobic residue; and X 11 is D; and iii) the EF hand motifs are linked by a 12 or 13 amino acid residue linker, each amino acid residue of the linker being a canonical amino acid, with the proviso that the third and fourth EF hand motifs are linked by the following sequence: (X) 5 -R-(X) 6 , each X is independently a canonical amino acid, and at least one amino acid in any linker is hydrophobic.

2. X in the first EF hand motif, the second EF hand motif, and / or the fourth EF hand motif 7 is independently T or S.

3. X in the third EF hand motif 4 The protein of claim 1, wherein is A.

4. X in the third EF hand motif 8 The protein of claim 1, wherein is L.

5. X in the third EF hand motif 10 is L, I or M.

6. 2. The protein of claim 1, wherein the protein comprises the following sequence: Or a protein having 70% identity to either SEQ ID NO:1 or SEQ ID NO:

2.

7. 2. The protein of claim 1, wherein the protein comprises the following sequence: Or a protein with 70% identity thereto.

8. The protein of claim 1 , wherein the protein is complexed with a rare earth element.

9. The protein of claim 1 , wherein the rare earth element is a light rare earth element.

10. The protein according to claim 8 , wherein the rare earth element is a heavy rare earth element.

11. 2. The protein of claim 1, wherein the protein comprises the following sequence: where X is any canonical amino acid residue other than R.

12. 12. The protein of claim 11, wherein the protein comprises the following sequence:

13. 2. The protein of claim 1, wherein the protein has the following sequence:

14. 2. The protein of claim 1, wherein the protein has the following sequence:

15. 1. A protein with enhanced REE / FREE selectivity, comprising a first EF hand motif, a second EF hand motif, a third EF hand motif, and a fourth EF hand motif, each EF hand motif comprising 11, 12, or 14 amino acid residues, wherein when the first EF hand motif, the second EF hand motif, the third EF hand motif, and the fourth EF hand motif each have 12 amino acid residues, each EF hand motif has the following sequence: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E where i) First and fourth EF hand motifs each X 1 is independently D or N; each X 2 are independently any canonical amino acid; each X 3 is independently D, N, or E; each X 4 are independently any canonical amino acid; each X 5 is independently D, N, or E; each X 6 are independently any canonical amino acid; each X 7 are independently any canonical amino acid; each X 8 are independently hydrophobic residues; each X 9 is independently D, E, or T; each X 10 are independently hydrophobic residues; and each X 11 are independently any canonical amino acid; ii) Regarding the second EF hand motif X 1 is N; X 2 is any canonical amino acid; X 3 is D; X 4 is any canonical amino acid; X 5 is D; X 6 is any canonical amino acid; X 7 is T or S; X 8 is a hydrophobic residue; X 9 is E; X 10 is any canonical amino acid; and X 11 is any canonical amino acid; and iii) Regarding the third EF hand motif X 1 is D; X 2 is any canonical amino acid; X 3 is D; X 4 is D; X 5 is D; X 6 is G; X 7 is T or S; X 8 is a hydrophobic residue; X 9 is D; X 10 is any canonical amino acid; and X 11 is any canonical amino acid iv) at least one X in the second EF-hand motif and at least one X in the third EF-hand motif 2 is P; and v) The EF hand motifs are linked by 12 or 13 amino acid residue linkers, where each amino acid in the linker is a canonical amino acid and at least one amino acid in any linker is hydrophobic.

16. 16. The protein of claim 15, wherein the protein has the sequence:

17. X in the second and / or third EF hand motif 8 is L, I, M, or V.

18. A device comprising the protein of claim 1 or claim 15.

19. 20. The device of claim 18, wherein the device is a filter, membrane, sensor, portable detector, plate reader, fluorometer, biosensor, or in-line monitor.

20. A kit comprising the protein of claim 1 or claim 15, or a device comprising the protein of claim 1 or claim 15.

21. 1. A method for isolating rare earth elements, comprising: A method for isolating rare earth elements, comprising contacting the protein of claim 1 with a sample containing rare earth elements, wherein the rare earth elements bind to one or more proteins of claim 1, thereby removing the proteins from the sample.

22. 22. The method of claim 21, wherein the sample is drinking water, wastewater, groundwater, ash pond, aqueous extract from contaminated soil, wastewater, leachate, aqueous extract or leachate from solid waste such as electronic waste, aqueous extract or leachate from ore or mine tailings, or a solid sample.

23. 22. The method of claim 21, wherein the one or more rare earth elements are lanthanides and are selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Sc, Y and ions thereof.

24. 22. The method of claim 21, further comprising detecting and quantifying one or more rare earth elements.

25. 22. The method of claim 21, wherein a plurality of different rare earth elements are bound to the protein.

26. 26. The method of claim 25, wherein each different rare earth element is individually separated from the protein.

27. A method for determining whether a light rare earth element is present in a sample, the method comprising contacting the sample with the protein of claim 1 and determining whether the protein forms a dimer.

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