Lanthanum modulin homologues with improved rare earth separation properties

Abstract

Proteins bound to rare earth metals are provided. The protein may have enhanced REE / REE selectivity. The protein can have four EF hand motifs, and each EF hand motif has 11, 12 or 13 amino acid residues. Each EF hand motif is separated by 12 or 13 amino acid residues, where each amino acid residue is any standard 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 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. Devices and kits comprising the proteins of the present disclosure are also provided. Methods of using the proteins and devices are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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.

[0003] Statement Regarding Federally Funded Research

[0004] 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.

[0005] Sequence Listing

[0006] This application contains a sequence listing, which is submitted in .xml format and is incorporated herein by reference in its entirety. This .xml copy was created on September 18, 2023, is named "074339_00251_ST26.xml" and is 114,140 bytes in size. Background Art

[0007] The irreplaceable role of rare earth (RE) elements in ubiquitous modern technologies, from permanent magnets to LEDs and fluorescent materials, has rekindled interest in one of the grand challenges of separation science: the efficient separation of lanthanides. Due to the similar physicochemical properties of the dominant +III ions of the 15 elements, their ionic radii range from La to III He Lu III Only decline between The separation of these 15 elements is complex, which also leads to the coexistence of these metals in rare earth element-containing ores. Conventional hydrometallurgical liquid-liquid extraction methods for producing RE utilize organic solvents (such as kerosene) and toxic phosphonate extractants and require dozens or even hundreds of stages to obtain high-purity individual RE oxides. The low efficiency and significant environmental impact of separating RE have stimulated research efforts on alternative ligands with greater separation factors between adjacent RE and greener process designs to achieve RE separation with fewer stages and using all-aqueous phase chemistry.

[0008] The discovery of the founding member of the lanmodulin (LanM) family of lanthanide-binding proteins demonstrates that nature has evolved macromolecules with superior selectivity to synthetic f-element chelators. The prototype LanM from Methylorubrumextorquens AM1 (Mex-LanM) is a small (12-kDa) monomeric protein that exhibits a selective conformational response to picomolar concentrations of lanthanides and actinides. This protein has advanced the understanding of lanthanide uptake in methylotrophs and serves as a technology platform for f-element detection, recovery, and separation. Unusually among RE chelators, Mex-LanM favors the larger and more abundant light REs (LREs), especially La III -Sm III , rather than heavy RE (HRE). Summary of the Invention

[0009] The present disclosure provides proteins that bind rare earth metals. Also provided are devices and kits comprising the proteins of the present disclosure. Also provided are methods of using the proteins and devices.

[0010] In aspects of the present disclosure, proteins that bind metals (e.g., lanthanides and / or actinides) are provided. Other metal binding proteins are disclosed in WO2020051274 and WO2023004333, which are incorporated herein by reference.

[0011] The proteins disclosed herein can have various lengths. For example, the proteins disclosed herein have between 65 and 160 amino acid residues, including all integer amino acid values ​​and ranges therebetween. For example, the proteins have a molecular weight of between about 8 kDa and 14 kDa, including all 0.1 Da values ​​and ranges therebetween (e.g., about 12 kDa). The proteins disclosed herein include at least one segment that can bind one or more rare earth metals. In various examples, the segment has at least 70% homology to the sequence of Hansschlegelia quercus LanM (which may be referred to as Hans-LanM), for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% homology. In other various examples, the proteins are truncated. For example, the protein is truncated at the N-terminus by deleting the first 10, 20, 30, or 40 residues of the complete translated sequence. In another example, the protein is truncated at the C-terminus by deleting the last 10, 20, 30, or 40 residues of the complete translated sequence. In various examples of truncated sequences, EF hands 2 and 3 and most of the hydrophobic core of the protein are retained.

[0012] In various examples, the protein or peptide of the present disclosure is capable of dimerization when in contact with a metal / metal ion, and the protein or peptide may comprise 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 (e.g., 12 amino acid residues). Each EF-hand motif is separated by 12 or 13 amino acid residues, wherein each amino acid residue is any standard 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, wherein each X is any standard amino acid residue. When the EF-hand motif has 12 amino acid residues, the motif may have the following sequence:

[0013] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E.

[0014] 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 Each independently represents any standard amino acid residue. In various examples, X of EF1, EF2 and / or EF4 7 Is T or S. For EF3, X 1 It is N; X 3 It is D; X 4 It is G or A; X 5 Is D or N; X 7 Is T or S; X 8 is a hydrophobic residue, X 9 It is E; X 10 is a hydrophobic residue; X 11 is D; and X 2 and X 6Each is independently any standard 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, EF3's X 8 is L. In various examples, EF3's X 10 is L, I, or M. Without intending to be bound by any particular theory, the dimer strength of this protein is believed to depend on the identity of the bound metal ion, with dimers preferentially formed 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.

[0015] In various examples, the proteins or peptides disclosed herein may have enhanced REE / REE selectivity. This selectivity may be between light rare earth metals and heavy rare earth metals and exceed the selectivity of M. extorquens lanthanum. Such a protein may comprise 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, and each EF-hand motif is separated by 12 or 13 amino acid residues, wherein each residue is a standard 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:

[0016] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E.

[0017] For the first EF-hand motif (ie, EF1) and the fourth EF-hand motif (ie, 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 、X4 、X 6 、X 7 and X 11 Each independently represents any standard amino acid residue. In various examples, X of EF1 and / or EF4 7 Is T or S. For EF2, X 1 It is N; X 3 It is D; X 5 It is D; X 7 Is T or S; X 8 is a hydrophobic residue; X 9 It is E; X 12 is E; and X 2 、X 4 、X 6 、X 10 and X 11 Each is independently any standard 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, IM or V. For EF3, X 1 It is D; X 3 It is D; X 5 It is D; X 6 It 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 Each independently is any 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 of EF2 or EF3 2 is P. A protein having this sequence can be linked to another protein of the present disclosure via a peptide linker described herein.

[0018] In aspects of the present disclosure, a device is provided. The device comprises one or more proteins of the present disclosure.

[0019] In aspects of the present disclosure, kits are provided. The kits may provide one or more proteins of the present disclosure and / or one or more devices of the present disclosure. The kits may include instructions for use of the proteins or devices.

[0020] In aspects of the present disclosure, various methods of using the proteins and / or devices of the present disclosure are provided. The methods of the present disclosure can be used to bind one or more lanthanides and / or actinides, or to detect and / or quantify the amount of one or more lanthanides and / or actinides.

[0021] The method of the present disclosure can be a method for detecting and / or quantifying the amount of one or more lanthanides and / or actinides in a sample. The method can include contacting the sample with one or more proteins and / or devices of the present disclosure. The contacted sample can then be exposed to light, and the exposed contacted sample thereby produces emitted light. The resulting emitted light result can then be compared with a known standard curve for a specific lanthanide or actinide. The concentration can be determined by this comparison. A known standard curve can be prepared based on the desire to detect and / or determine the amount of any specific lanthanide or actinide. Methods for preparing standard curves are known in the art.

[0022] Methods using proteins and / or devices of the present disclosure can be methods for binding one or more rare earth metals (e.g., lanthanides and / or actinides) in a sample. The 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 of contaminated soil, drainage water (e.g., mine drainage water, such as acid mine drainage water), or leachate (e.g., electronic waste leachate or ore leachate). In various other examples, the sample is a solid sample. The method can be applied to samples of various pH values. For example, the sample has a pH of 6 or less, such as 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.

[0023] Various lanthanides (e.g., lanthanide ions) and / or actinides (e.g., actinide ions) can 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 an ion thereof. The bound lanthanides and / or actinides can be the same or different. The concentration of the lanthanides and / or actinides in the sample can be less than 1 ppm. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] For a fuller understanding of the nature and purpose of the present disclosure, reference should be made to the following examples and the accompanying drawings therein.

[0025] Figure 1 Hans-LanM differs from Mex-LanM in terms of sequence and RE / RE selectivity. A, Sequence similarity network (SSN) of LanM core sequences indicates that Hans-LanM forms a distinct cluster. The SSN includes 696 LanM sequences connected by 48,647 edges, with a BLAST E-value threshold of 1×10 -5 The sequence identity threshold is 65%. The black box surrounds the nodes clustered with Hans-LanM. The LanM sequences associated with Mex (▼) and the four sequences within Hansschlegelia (▲) are enlarged compared to the other nodes (○). The color of the node represents the family from which the sequence originated. B, Comparison of the sequences of the four EF hands of Mex- and Hans-LanM. C, Comparison of the sequences of the four EF hands of Hans-LanM and La III Circular dichroism spectra of a representative titration showing the metal-associated conformational response to increased helicity; apoprotein is bold black, La III Saturated proteins are bold red. D, Hans-LanM and La III 、Nd III and Dy III CD titration (pH 5.0). Each point represents the mean ± sd from three independent experiments. e, K of Mex-LanM and Hans-LanM d,app Comparison of pH values ​​(pH 5.0), plotted against ionic radius. Mean ± sem from three independent experiments.

[0026] Figure 2 The dimerization equilibrium is shown to be sensitive to LRE vs. HRE or non-RE coordination. a, Apparent molecular weight of Hans-LanM complex with RE as determined by analytical SEC (solid line) or SEC-MALS (dashed line). Conditions are shown in Table 1. Each individual data point is the result of a single experiment. b, La as determined by X-ray crystallography III Bound Hans-LanM dimer. La III Ions are spheres, Na I Gray spheres. c, Detailed view of the dimer interface near EF3 of chain A (cartoon). Arg100 from chain C (cartoon) anchors Asp93 of chain A and two EF3 La IIIHydrogen bonding network of the ligands (Glu91 and Asp85). These interactions constitute the only polar contacts at the dimer interface, providing a means to control the radius of the lanthanide binding site at EF3. d, Schematic diagram of the interactions at the dimer interface. Dashed lines indicate hydrogen bonding interactions, while other dashed lines indicate hydrophobic contacts. e, La from SAXS data set III Combined (left) and Dy III DENSS projection of the electron density of the combined (right) Hans-LanM and the PyMOL generated dimer La III - Ribbon diagram superposition of the Hans-LanM crystal structure.

[0027] Figure 3 Shown is the use of an extended hydrogen bonding network by Hans-LanM to control lanthanide selectivity. a, La III -Magnified view of EF2 and EF3 in Hans-LanM. La III Ions are shown as green spheres. Coordination bonds and hydrogen bonds are indicated by dashed lines. Residues contributed by the A chain are shown, and (in the case of EF3) residues contributed by the C chain are also shown. (Inset) La III -Hans-LanM and Dy III -Hans-LanM superposition shows the carboxyl group 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.

[0028] Figure 4 The full utilization of Hans-LanM for Nd / Dy separation in a single-stage process is shown. a, Hans-LanM and the R100K variant show greater differences in Nd vs. Dy complex stability upon citrate desorption compared to Mex-LanM. Mean ± sem of three independent experiments. III [Citrate] 1 / 2 The significant difference (**) between the two groups shows that the dimerization of La III Effect of complex stability (p < 0.01, ANOVA with Bonferroni post hoc test). Mex-LanM Nd and Dy data are from Dong et al., ACS Cent. Sci. 7, 1798, 2021. b, Fluorescence spectra titration of Hans-LanM and R100K variants at pH 5.0 (λ ex =280nm,λ em=333 nm), which depicts the desorption of malonate-induced metal:protein 2:1 complexes. Mean ± sem of three independent experiments (except R100K, which is a single experiment for each condition). c, Comparison of the distribution factors of immobilized Hans-LanM, R100K-Hans-LanM, and Mex-LanM (pH 5.0, approximately 0.33 mM each RE, La III -Dy III Each point represents the mean ± SD of three independent experiments. d, Separation of a 95:5 Nd:Dy mixture using immobilized R100K-Hans-LanM and a three-step concentration protocol of malonic acid followed by desorption with pH 1.5 hydrochloric acid. One bed volume was 0.7 mL.

[0029] Figure 5 The sequence alignment of Mex-LanM and Hans-LanM (after removing the signal peptide) is shown, which shows 33% sequence identity. The EF hands are shown in bold. The full-length Hans-LanM sequence including the predicted signal peptide is shown in Table 12. The Hans-LanM protein used in this study consists of residues A24-K133. The sequence is shown as

[0030]

[0031] Figure 6 Shown is the relationship between Hans-LanM (15 μM) and La monitored by CD spectroscopy. III and Dy III Stoichiometric metal titration of . Molar ellipticity at 222 nm is plotted against the amount of metal added. Experimental conditions: 20 mM acetic acid, 100 mM KCl, pH 5.0. Each data point is the mean ± SD of two independent measurements. Figure 1 These observations suggest that Hans-LanM and RE III The following model of ionic interactions: tight (albeit preferentially LRE) binding of the metal to one site leads to a conformational change; the second site responds cooperatively with the first site to the LRE to give a full conformational response, but it responds non-cooperatively and only at concentrations >0.5 μM to Dy; and the third site does not induce an observable conformational change with any RE. Based on previous work on Mex-LanM and the crystal structure described here, the third site is hypothesized to be EF1; EF2 and EF3 cannot be clearly distinguished as the first and second sites. This more complex response profile than that of Mex-LanM appears to be tuned to ensure a fully cooperative response only to LREs.

[0032] Figure 7Shown are apo Hans-LanM (black) and the reaction with 3.0 equivalents of La III Size exclusion chromatogram of metallated Hans-LanM (red). The S75 column volume was 24 mL. As with apo-Mex LanM, the apoprotein eluted over a broad molecular weight range (30-70 kDa), indicating the presence of multiple disordered conformations. III The bound protein showed a soluble high molecular weight species formed when RE was added to Hans-LanM at high concentrations, and a symmetrical peak at approximately 28 kDa indicating the presence of a dimer.

[0033] Figure 8 shows RE III Size exclusion chromatograms of Hans-LanM complexes (RE = La, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Y). Apo-Hans (590 μM, 100 μL) was metallated with 3.0 equivalents of RE, 0.5 equivalents at a time, mixed, and loaded onto a 24 mL calibrated analytical S75 column as described. a, The apparent molecular weights shown in the chromatogram for RE = La-Dy indicate the presence of Hans-LanM dimers of La and Nd, which gradually shift toward lower apparent molecular weights, possibly indicating a decreasing proportion of dimers in rapid equilibrium with monomers. b, With the exception of Dy, the species formation of the complex is mixed but suggests the presence of a more extended monomer or a slowly exchanging dimer population.

[0034] Figure 9 SEC-MALS traces of La-, Nd- and Dy-bound Hans-LanM are shown, which illustrate that the Dy complex elutes later and has a lower weight-average molar mass (see Table 3). Apo-Hans-LanM was mixed with 3 equivalents of each RE III ion incubation, precipitates and aggregates were removed by centrifugation and analytical SEC, and the protein was injected onto the column at a concentration of 114-128 μM.

[0035] Figure 10 ITC tracking of dimer dissociation of apo Hans-LanM is shown. (Top) Representative ITC trace for a 300 μM titration of protein into buffer. (Bottom) Thermogram derived from the above data fitted to a dimer dissociation model using NanoAnalyze software, with parameters presented in Table 18. Conditions: 30 mM MOPS, 100 mM KCl, pH 7.0, and 30°C.

[0036] Figure 11 Shows Dy tracked by ITC IIIDimer dissociation of 2-Hans-LanM. (Top) Representative ITC trace of 300 μM protein titrated into buffer. (Bottom) Thermogram derived from the above data fitted to a dimer dissociation model using NanoAnalyze software, with parameters presented in Table 18. Conditions: 30 mM MOPS, 100 mM KCl, pH 7.0, and 30°C. Using Equation 1, Dy can be calculated under SEC-MALS conditions. III -Concentrations of Hans-LanM monomer and dimer, giving [P] = 18.9 μM (obtained from Table 17) and K from ITC 二聚体 =60 μM (Table 18). We obtained [M] = 13.5 μM and therefore [D] = 3.3 μM. Since [D] is about 25% of [M], this result corresponds well to the average mass of 15.5 kDa obtained by SEC-MALS for this form, which is about 25% higher than the expected MW of 11.9 kDa, indicating the presence of partial dimerization. This in turn supports our interpretation that the SEC-MALS results indicate a rapid monomer-dimer equilibrium in which individual monomers and dimers cannot be resolved, and therefore appear as a weighted average of the two populations.

[0037] Figure 12 Shows La tracked by ITC III Titration of 2-Hans LanM. (a, top) Representative ITC trace of a 150 μM protein titration into buffer. (a, bottom) Thermogram derived from the above data. (b, top) Representative ITC trace of a 540 μM protein titration into buffer with an initial 0.2 μL injection followed by 9 × 5.0 μL additions. (b, bottom) Thermogram derived from the above data. No difference in heat between each injection was observed in these experiments, further reinforcing the hypothesis that La III The results show that the bound dimer is very tightly bound. Conditions: 30 mM MOPS, 100 mM KCl, pH 7.0, and 30° C. We can estimate La by using Equation 1 (based on the peak concentration from SEC-MALS [P] = 18.4 μM (Table 17)) and conservatively assuming a minimum threshold for observable monomer [M] of 10% of the total protein concentration (1.84 μM), as it would appear in SEC-MALS as a difference of approximately 1 kDa from the theoretical monomer MW of 11.9 kDa (this difference is observed in the case of apo-Hans-LanM, see Table 17). III - the maximum dimer dissociation constant of Hans-LanM. In this case, solve for K 二聚体 0.4 μM was obtained, which represents La IIIThe maximum dissociation constant of the bound protein dimer. III Compared with the presence of Dy III In this case, Hans-LanM exhibited more than 100-fold enhanced dimerization.

[0038] Figure 13 Comparison of the topological structures and key residues of Hans-LanM and Mex-LanM is shown. a, La III -Hans-LanM and Nd III -Mex-LanM topology diagram. Despite having 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 and decorated with two auxiliary helices (i.e., EF1 and EF3 in front). Figure 32 As explained, the presence of Nd in EF4 of Mex-LanM III ions are a result of the high protein concentration used for crystallization. b, Sequence alignment of key regions of Hans-LanM, Mex-LanM, and H. sapiens calmodulin. Similarities in metal-binding residues in these proteins are highlighted in light blue; note the presence of a Glu residue at position 9 of each EF hand in Hans-LanM, which is unique relative to other proteins. Residues involved in interactions at the dimer interface in Hans-LanM are shown in bold (hydrogen bonding interactions: E91, D93, R100) (hydrophobic interactions: I43, I47, H48, T63, M92, L96). The charge of the residues corresponding to Hans-LanM D93 and R100 is reversed in Mex-LanM (K94, E101). Several of the residues involved in hydrophobic interactions in Hans-LanM (I43, I47, L96) correspond to similar residues in Mex-LanM (L44, L48, L97), while other residues (H48, T63, M92) correspond to residues that lack bulky side chains (A49, G64) or are charged (K94). This analysis also highlighted another potentially important difference, namely that calmodulin possesses 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 position. In contrast, the Hans-LanM and Mex-LanM EF hands have a glycine at only one of these positions (or no glycine in Hans-LanMEF1). The sequence shown is

[0039]

[0040] Figure 14 SAXS raw data of La-, Nd-, and Dy-bound Hans-LanM are shown. In-house RigakuBioSAXS2000 nano SAXS data sets were collected on SEC-MALS fractions in the presence of different metal ions in 30mM MOPS, 100mM KCl, 5% glycerol, pH 7.0. a, La-bound protein was 1.5mg / mL, Nd-bound protein was 1.4mg / mL, and Dy-bound protein was 1.6mg / mL. A 60-minute SAXS data set (with 6 10-minute images) was collected using an automatic sampling quartz flow cell. Buffer SAXS data were collected over 60 minutes using the same flow cell and used for reference subtraction. The superposition of six 10-minute images averaged in each case shows that there is no X-ray radiation damage. b and c, La and Dy SAXS data were fitted with CRYSOL (ATSAS) of the crystallographic La-Hans-LanM and Dy-Hans-LanM dimer models. Table 6 lists the chi-square values.

[0041] Figure 15 Shown are superpositions of the Guinier plots of La-, Nd-, and Dy-bound Hans-LanM. The fitted radius of gyration (R g )for and (See Table 4). Note that the R g The differences between the values ​​are smaller than what can be expected based on SEC-MALS R g The values ​​(Table 17 shows that the hydrodynamic radius of La / Nd vs. Dy is about The difference is not significant because the protein concentration used in SAXS is 5 times higher than that in SEC-MALS and therefore the population of Dy-bound dimers is significantly larger in SAXS experiments. Nevertheless, these RE-dependent differences in SAXSR g The value is within the uncertainty range.

[0042] Figure 16 Shows La III -bound Hans-LanM solvent encapsulation. Density from Solution Scattering (DENSS) is an algorithm that calculates ab initio electron density maps from solution scattering data. The DENSS electron density map is shown as a transparent surface in a PyMOL generated representation of the dimer La. III -Hans-LanM crystal structure superposition (here colored by chain (light blue and green)). La IIIIons are represented as gold spheres. The color gradient is from lowest electron density to highest electron density: blue (2σ) to cyan (5σ) to green (7.5σ) to yellow (10σ) to red (15σ). Encapsulation and manual fitting of the model were performed in PyMOL. The CRYSOL fit of the calculated SAXS curve was superimposed on the experimental SAXS curve with a chi-square fit of 1.1 (Table 6). Figure 2 La shown in e III -Hans-LanM data are the same.

[0043] Figure 17 Shows Nd III Solvent encapsulation of bound Hans-LanM. DENSS electron density map with dimer La shown as a transparent surface in a PyMOL-generated representation. III -Hans-LanM crystal structure superposition (here colored by chain (light blue and green)), where La III Ions are represented as gold spheres. Encapsulation and manual fitting of the model were performed in PyMOL. The CRYSOL fit of the calculated SAXS curve was superimposed on the experimental SAXS curve with a chi-square fit of 1.3 (Table 6).

[0044] Figure 18 Shows Dy III Solvent encapsulation of bound Hans-LanM. DENSS electron density map with dimer La shown as a transparent surface in a PyMOL-generated representation. III The -Hans-LanM crystal structure was weakly superimposed. The CRYSOL fit of the calculated dimer SAXS curve was superimposed with the experimental SAXS curve, with a chi-square fit of 3.8 (Table 6).

[0045] Figure 19 Shown is a distance distribution (P(r)) analysis showing that Hans-LanM undergoes a transition from a single species with La and Nd to two states with Dy. Superposition of the pairwise distance distribution functions P(r) for the La- (green), Nd- (blue), and Dy-bound (red) Hans-LanM data sets. R from this analysis g value( (La), (Nd) and (Dy)) and R obtained from Guinier analysis g The values ​​are similar. For La and Nd complexes, P(r) has a bell-shaped shape, representing scattering from spherical particles. In the case of Dy, P(r) shows three shoulders, indicating the presence of a mixture of species (e.g., a monomer / dimer mixture).

[0046] Figure 20 Kratky plots are shown, indicating that the flexibility of Hans-LanM increases from La to Nd to Dy. Kratky plots of La- (green), Nd- (blue), and Dy-bound (red) Hans-LanM. Kratky plots derived from SAXS data qualitatively inform about the flexibility and / or folding of the protein. The progressive deviation from the q-axis from the La- to Nd- to Dy-bound forms indicates an increase in disorder, which may be related to the weaker Dy III Binding is associated with reduced cooperativity ( Figure 1 d) and / or is related to the monomer-dimer equilibrium. The unit of q is

[0047] Figure 21 Shows La III Combined Hans-LanM EF hand 3 and 4 2F o -F c Electron density map (grey grid, outlined at 1.0σ) and anomalous difference map (purple grid, outlined at 3.0σ). a, In EF4, the solvent is clearly indicated, and the absence of anomalous difference density shows that La III There is no placeholder for this position. Figure 52 For the reasons described in , the metal ions were modeled as fully occupied Na I b, In EF3, representing EF hands 1–3, the electron density map indicates no solvent coordination, and the anomalous difference density map is consistent with the fully occupied La III Ions consistent.

[0048] Figure 22 Structural comparison of the LanM EF hand with the EF hand from calmodulin (CaM) and with lanthanide-dependent methanol dehydrogenase (MDH). a, from La III Combined Hans-LanM EF2. La III Ions are green spheres. b, from Nd III EF3 of Mex-LanM bound. Nd III Ions are light green spheres and coordinating solvent molecules (w1 and w2) are red spheres. c, EF2 from Homo sapiens CaM (PDB code: 1CLL). Ca II Ions are grey spheres and coordinating solvent molecules are red spheres. d, from La III Lanthanide-dependent MDH active site XoxF( resolution, PDB code: 6DAM). 10-coordinated La IIIThe ions are shown as green spheres. The enzyme also requires the pyrroloquinoline quinone cofactor (PQQ). In addition to the D5 residue being monodentate in CaM (Asp24) and bidentate in Mex-LanM (Asp88), and the presence of an additional water molecule (w1) in Mex-LanM, Nd III -Mex-LanM and Ca II -CaM have nearly identical major coordination spheres. These images also highlight the consequence of Hans-LanM having an Asn (N1) at position 1 of the EF hand versus Asp (D1) in Mex-LanM and CaM. 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 is hydrogen bonded to the backbone CO of the Asp62 residue (5th position), whereas in Mex-LanM and CaM the non-coordinating atom is an oxygen, allowing hydrogen bonding to the backbone NH of the residue at position 6. This difference is accommodated by Hans-LanM having a Gly at position 4 in EF hands 2 and 3, whereas Mex-LanM has a Gly at position 6, and CaM has a Gly residue at both positions ( Figure 13 b).

[0049] Figure 23 The Horrocks method is used to show the III Spectroscopic estimation of the number of coordinating solvent molecules (q) in 2-Hans-LanM. Eu III The luminescence lifetime of the complex is empirically related to q. Triangle: value of the luminescence decay time constant (τ, in ms, left y-axis). By comparison, τ of Hans-LanM H2O = 1.24ms, while Mex-LanM is only 0.404ms. Circle: 1 / τ value (in ms) -1 , right y-axis). The fitting equation of 1 / τ versus the mole fraction of D2O was used to determine q. The uncertainty of q was taken as ±0.5. The q value of 0.11 is consistent with the absence of a 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 two independent samples.

[0050] Figure 24Shown are the extended hydrogen-bonding networks in the metal-binding sites of Hans-LanM and Mex-LanM. a, The metal site of Hans-LanM exhibits extensive hydrogen bonding 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 adjacent monomer. b, The metal site of Mex-LanM features a similar hydrogen-bonding pattern, but with solvent molecules w1 and w2 replacing Glu69. c, Zoomed-in view of the backbone flip, which allows hydrogen bonding interactions between the Asp and Asn residues at position 1 and the backbone amide and carbonyl groups in Mex-LanM and Hans-LanM, respectively.

[0051] Figure 25 The CD spectroscopy of R100K-Hans-LanM and La III 、Nd III and Dy III The fitting parameters are summarized in Table 7. III and Dy III The results (K d,app and molar ellipticity) were basically the same as those obtained with the wild-type Hans-LanM protein; III , a 2-fold weaker K d,app Conditions: 15 μM protein, 20 mM acetic acid, 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.

[0052] Figure 26 As apoprotein and 3 equivalents of La III SEC-MALS trace of metallated R100K-Hans-LanM. This apoprotein is similar to Mex-LanM and wild-type Hans-LanM ( Figure 7 ) migrate similarly, indicating disordered proteins. III The complex migrated as a single sharp peak. Both samples had similar weight-average molar masses as determined by MALS corresponding to the monomer (see Table 8). Conditions: 3 mg / mL protein, 30 mM MOPS, 100 mM KCl, pH 7.0.

[0053] Figure 27Shown is the LanM of mistletoe Hansschlegelia quercus and the LanM protein that predicts that can dimerize that is different from other LanM in phylogeny.Under the strict clock of minimum sequence length being 106 amino acids, the site isomorphism model based on the Whelan and Goldman matrix with invariant sites and four different gamma categories (WAG+I+Γ4) is used to construct the Bayesian phylogenetic diagram.The monophyletic group of the member that comprises Hans group is highlighted in gray.Node value indicates the posterior probability based on 10,000,000 iterations, wherein the pre-burn-in period is 25%.The scale bar represents 0.1 change per amino acid position.The LanM core sequence alignment for constructing phylogenetic diagram is colored with the Zappos scheme.The four EF hand domains are marked with lines at the bottom of the alignment, and the residues associated with dimer interaction are marked with asterisks. The Bayesian phylogeny constructed from this alignment supports the network structure, with the Hans taxa represented as monophyletic groups and more distant from the other sequences. Furthermore, the topology of the Hans taxa in the phylogenetic tree corresponds to the proximity seen in the network ( Figure 1 a and Figure 50 ). In this alignment, R100 is at position 87, marked with 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 Hans group LanM, indicating that these proteins all form dimers. Only a single LanM outside the Hans group (unclassified Hyphomicrobiaceae) has an Arg residue at position 87. The EF3 sequence in this ortholog lacks several Ln III ligand, but EF2 mediates the interaction with Arg in Hans (in EF3) 11 The presence of cysteine ​​residues at position 87 was characteristic, suggesting that this uncharacterized LanM may dimerize along a unique interface. Aside from the sequences included in this alignment, no sequences contain a basic amino acid at position 87—in contrast, most sequences (such as Mex-LanM) have an acidic residue (usually Glu). Furthermore, a group of LanMs containing two cysteine ​​residues was identified, one near each terminus (near EF1 and EF4). Given that LanM is a periplasmic protein, we propose that these proteins possess disulfide bonds between these residues, which may provide additional structural stability to LanM, but more evidence is needed to determine the role of these residues.

[0054] Figure 28 Shows Dy III -Two views of Hans-LanM's EF hand 3, of which 2F o -F cElectron density map (grey mesh, outlined at 1.0σ) and anomaly difference map (purple mesh, outlined at 3.0σ). a) Density associated with the metal ligand is clear, and no coordinated solvent is apparent. b) The hydrogen bonding network between EF3 and Arg100 of the adjacent monomer is also clearly visualized.

[0055] Figure 29 The X-ray absorption edge of Dy-Hans-LanM detected by fluorescence excitation is shown.

[0056] Figure 30 Shown for Dy III -Dy on Hans-LanM crystals III Anomalous diffraction data set collected at the edge (7793.5 eV) supports the assignment of the bound lanthanide as the HRE, Dy. Anomalous difference electron density maps are shown in purple grid (outlined at 4.0σ) for a representative metal binding site in chain A. In all four EF hands, we observed L III The peaks of the abnormal difference electron density maps above the edge are significantly stronger (Table 10). Interestingly, EF2 and EF3 show the largest abnormal difference map peaks, perhaps reflecting the biochemical observation of only two high affinity sites in the complex ( Figure 1 d, Figure 6 ).

[0057] Figure 31 Shows Dy III All four copies of the Arg100-EF3 hydrogen bonding network in the asymmetric unit of Hans-LanM show the same shift towards monodentate coordination in Glu91 and a shift of the hydrogen bond between this residue and Arg100 from La to III -Hans-LanM in about To Dy III -Hans-LanM One of the hydrogen bonds between Arg100 and Asp93 is also extended from La III -Hans-LanM Extended to Arg100-Asp85 hydrogen bond from La III -Hans-LanM Slightly compressed to Dy III -Hans-LanM Although it is possible that forced dimerization under the high concentration conditions of crystallography may alter the interactions between the monomers relative to those at low concentrations in solution, the La- and Dy-bound structures illustrate how carboxyl shifts of Glu91 can alter this second sphere hydrogen bonding network (which would appear to disfavor dimerization), an interpretation strongly supported by the characterization of the R100K variant (Table 8, Figure 25-27 ).

[0058] Figure 32 Shows Nd III The X-ray crystal structure of the bound Mex-LanM with a resolution of a, Overall structure of Mex-LanM. Due to the crystallization conditions (3.5 equivalents of Nd III and millimoles of protein), Nd in EF4 III ions are present; previous biochemical analyses have shown that the weakest binding equivalent is micromolar K d And this weak site is associated with EF4. b, 2F of EF3 o -F c Electron density map (grey grid, outlined at 1.0σ) and anomaly difference map (purple grid, outlined at 3.0σ) showing the relationship between the electron density and the Nd III Ion coordinated to two solvent molecules. Nd III Ions are light green spheres, while solvent molecules are red spheres. c, Details of the four EF hands. The metal coordination in EF1-3 is the same, where D1, D3, and T7 have a monodentate ligand as the backbone CO, and D5 and E 12 It is a bidentate ligand, and two water molecules (w1 and w2) provide nine-fold coordination. In EF4, the D3 residue (Asp110) is bidentate. Because EF4 possesses an Asn instead of an Asp at position 1, the non-coordinating side chain N cannot hydrogen bond with the backbone, which may contribute to the lower affinity for this site.

[0059] Figure 33 Shows La III -Hans-LanM and Nd III Comparison of the hydrogen bonding network connecting the metal binding site to the exiting helix in Mex-LanM. These extensions of the backbone CO-HN hydrogen bonding network within each helix including the metal site may contribute to RE III -LanM complex. a, In La IIIIn -Hans-LanM, although the E9 residue is a bidentate ligand and thus cannot undergo direct similar hydrogen bonding, Glu91 in EF3 is connected to the first backbone NH of the exit helix via a hydrogen bonding network involving Arg100 and Asp93. In the presence of HRE, disruption of this network and, therefore, disruption of the connection between the helix and the metal site may also contribute to the lower stability of the HRE-Hans-LanM complex. b, In the presence of Nd III In -Mex-LanM, the D9 residue (Asp92 in EF3) directly connects Glu95NH from the exit helix to the Nd III Coordinating solvent (w2). The different lengths of these two hydrogen bonds caused by the coordination of different REs may contribute to the selectivity trend in Mex-LanM.

[0060] Figure 34 Shown from La III Representative fluorescence emission intensity and wavelength (λ) of a single measurement of wild-type Hans-LanM during the titration max ) changes. a, Hans emission spectrum (λ ex =278nm). La III Binding increases the intensity by 2-fold and shifts the λmax from 343 nm to 333 nm. b, Excitation and emission wavelengths indicate that two Trp residues (Trp79 and Trp95) near EF2 and EF3 of this protein make the main contribution to the spectrum. Trp95 is aligned with Tyr96 of Mex-LanM ( Figure 5 ), whose fluorescence intensity has been shown to be also sensitive to metal binding and / or associated protein conformational changes. Conditions: 20 μM protein, 30 mM MOPS, 100 mM KCl, pH 7.0.

[0061] Figure 35 Representative Nd leakage curves for column-immobilized Hans-LanM and R100K-Hans-LanM are shown. The experiments used 0.4 mM Nd in 7 mM homo-PIPES at pH 5.0. III. For Hans-LanM, 4.4±0.08 μmol / mL protein was immobilized, with a Nd adsorption capacity of 4.6±0.23 μmol / mL (1.06 equiv). For R100K-Hans-LanM, 3.2±0.06 μmol / mL protein was immobilized, with a Nd adsorption capacity of 6.66±0.33 μmol / mL (2.08 equiv). Each protein was immobilized once; the uncertainty of the immobilized protein represents the s.d. from triplicate protein concentration determinations by BCA assay, and the uncertainty in the adsorption capacity was assumed to be 5% based on our previously reported Lan column experiments.

[0062] Figure 36 The Hans complex with the HRE is shown to be more easily precipitated than the LRE complex. (A) Hans + Dy (left) and La (right), (B) the sample from (A) after centrifugation, and (C) the sample from (A) after reconstitution with EDTA. This property may be amenable to separation procedures.

[0063] Figure 37 Shown is the apparent K of 15 μM Hans-LanM compared to His-tagged Mex-LanM monitored using CD spectroscopy. d Buffer: 30mM acetic acid, 100mM KCl, 0-10mM Ln III , 10 mM EDTA or EGTA, pH 5.0.

[0064] Figure 38 The CD spectrum of Mex-LanM I42L / N108D / I115L (10 μM) is shown, which shows that the protein exhibits additional α-helical content relative to the wild-type protein in the apo state (left). III CD spectra of Mex-LanM I42L / N108D / I115L (5 μM) (right) Buffer: 20 mM acetic acid, 100 mM KCl, 0-10 mM Nd III , 10 mM EDTA, pH 5.0. Note that there is some inaccuracy in the ellipticity due to the low concentration of the single accumulation, but the data indicate that K d,app The binding stoichiometry of wild-type Mex-LanM and its variants was determined (bottom). Nd was added as previously described (Cotruvo et al., JACS 2018). III Titrate into a solution of protein and xylenol orange. The absorbance at 574 nm is relative to that of Nd III Equivalent drawing of .

[0065] Figure 39 The free Nd in various EDTA buffers was shown using CD spectroscopy. III CD titration curve of 20 μM Ex-LanM (A32D / A117K) monitored by ion concentration (left) and [θ] 222nm (Right). Buffer: 30 mM acetic acid, 100 mM KCl, 0-10 mM Nd III , 10 mM EDTA, pH 5.0. Apparent K d is 20±1pM, where n=1.85±0.08. d,app and below K d,app The point is at the lower end of the EDTA buffer range (less than the "% high solubility" value of 2%, or 200 μM total Nd), and metal binding to 20 μM protein may affect the free metal concentration but is not accounted for in the calculation. Therefore, this experiment slightly overestimates the true K d,app value (i.e., the affinity is underestimated).

[0066] Figure 40 The free Nd in various EDTA buffers was shown using CD spectroscopy. III CD titration curve of 20 μM Ex-LanM (A32D / A117R) monitored by ion concentration (left) and [θ] 222 nm (right). Buffer: 30 mM acetic acid, 100 mM KCl, 0-10 mM Nd III , 10 mM EDTA, pH 5.0. Apparent K d is 22±1pM, n=1.51±0.08. d,app and below K d,app The point is at the lower end of the EDTA buffer range (less than the "% high solubility" value of 2%, or 200 μM total Nd), and metal binding to 20 μM protein may affect the free metal concentration but is not accounted for in the calculation. Therefore, this experiment slightly overestimates the true K d,app value (i.e., the affinity is underestimated).

[0067] Figure 41 Shown is the relationship between the III Comparative time-resolved (left) and steady-state (right) fluorescence emission spectra of bound LanM_001 (Mex-LanM) and LanM_002 (Hans-LanM). [Protein] = 20 μM, [Metal] = 60 μM. Buffer: 30 mM MOPS, 100 mM KCl, pH 7.0. ex =280nm, delay = 150μs (left). ex =280nm (right).

[0068] Figure 42 Shows the relationship between Tb III Comparative time-resolved (left) and steady-state (right) fluorescence emission spectra of bound LanM_001 and LanM_002. [Protein] = 20 μM, [Metal] = 60 μM. Buffer: 30 mM MOPS, 100 mM KCl, pH 7.0. ex =300nm, delay = 100μs (left). ex =280nm (right).

[0069] Figure 43 Shows the relationship between Sm III (Left) and Dy III (Right) Comparative time-resolved fluorescence emission spectra of bound LanM_001 and LanM_002. [Protein] = 20 μM, [Metal] = 60 μM. Buffer: 30 mM MOPS, 100 mM KCl, pH 7.0. ex =280nm, delay = 150μs (left). ex =290nm, delay =125μs (right).

[0070] Figure 44 The results show that 20 μM LanM_012 in the presence of 4.0 equivalents of La III or Dy III Size exclusion chromatography (left) under the conditions of 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0. Size exclusion chromatography (right) of 400 μM LanM_012 in the absence of lanthanides. The peak at a retention volume of approximately 14.0 mL indicates the presence of 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.

[0071] Figure 45 LanM_012, monitored at 575 nm, tightly binds 2.0 equivalents of La in competition with the indicator xylenol orange. III Protein: 20 μM Buffer: 20 mM MES, 100 mM KCl, 5 mM acetic acid, pH 6.0.

[0072] Figure 46 The expression of La to 20 μM LanM_012 at pH 5.0 was followed using circular dichroism spectroscopy. IIITitration. The spectrum of the apo protein (0 equivalents) indicates the presence of a well-folded protein with a high α-helical content. The addition of metal results in a small shift in the shape of the curve around 205 nm, indicating ordering of the loop regions and suggesting only minor changes in the overall secondary structure of the protein. These preliminary results indicate that the apo form of the protein is well-folded. Buffer: 20 mM acetic acid, 100 mM KCl, pH 5.0.

[0073] Figure 47 CD spectra of LanM_012 in apo form or in complex with 2.0 equivalents of La or Dy are shown. The temperature was increased at 2°C / min, and spectra were collected after each increase. The spectra of Apo (A), La (B), and Dy (C) at 16°C, 28°C, 74°C, and 84°C were plotted. The signals between 218nm and 222nm for each obtained spectrum were averaged and plotted against temperature (D). The dotted line indicates the ellipticity of the apoprotein at 222nm at high temperature. Protein: 20 μM. Buffer: 30mM MOPS, 100mM KCl, pH 7.0.

[0074] Figure 48 Shown is the tryptophan fluorescence titration of 10 μM LanM_012 followed in the presence of various free metal concentrations (left). Buffer: 20 mM acetic acid, 100 mM KCl, 0-10 mM Ln III , pH 5.0. 10 mM EDTA and 10 mM EGTA were used to remove free La. III and Dy III Concentration is buffered. ex = 280 nm. LanM_012 (20 μM) was metallated with 3.0 equivalents of La, Nd, or Dy, and tryptophan fluorescence was monitored at 333 nm in the presence of various citrate concentrations (right). Dissociation of the protein-metal complex results in a loss of tryptophan fluorescence intensity. Buffer: 20 mM acetic acid, 100 mM KCl, pH 5.0. ex =280nm.

[0075] Figure 49 Shows the Sm III (A) and Dy III (B) Time-resolved fluorescence emission spectra of apo LanM_012 compared with LanM_012-Ln2. III (C) and Tb III(D) Comparative steady-state fluorescence emission spectra of apo LanM_012 and LanM_012-Ln2. [Protein] = 20 μM, buffer: 30 mM MOPS, 100 mM KCl, pH 7.0. ex =280nm, delay = 150μs(A); λ ex =290nm, delay = 125μs(B); λ ex =280nm(C);λ ex =280nm(D).

[0076] Figure 50 Shown from Figure 1 An expanded view of the inset of a (Hans cluster), including 20 sequences and 190 edges. The Hans cluster includes LanM from bacteria in the genera Hansschlegelia, Ancylobacter, Methylopila, Oharaeibacter, Starkeya, and Xanthobacter. Although these genera are restricted to this cluster, family-level members are scattered throughout the network, including one Xanthobacteraceae and 42 Methylocystaceae.

[0077] Figure 51 Shown are Hans-LanM and (a) Ca II (b) Nd III and (c)Dy III CD titration of a chelating agent buffer solution. III and La III The conformational change caused by Dy III (up to 0.3 μM) and Ca II (up to 5.5 mM) both induce similar, incomplete conformational changes in the protein. 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. The data in b and c are representative titrations from the three data sets used to generate the plot in the left panel. Figure 1 Representative titrations of three datasets plotted in d. Conditions: 15 μM protein, 20 mM acetic acid, 100 mM KCl, 10 mM EDTA (for Ca and Nd titrations) or EGTA (for Dy titrations), 0–10 mM metal ion. Each data point in (a, left) is the mean ± sd of three independent measurements.

[0078] Figure 52 Shows La III - X-ray crystal structure of the combined Hans-LanM at a resolution of a, Overall structure of the asymmetric unit, which consists of two Hans-LanM dimers and two citrate molecules from the crystallization solution. The structure of each monomer of the dimer is similar to that of Y III The NMR solution structure of the bound Mex-LanM is consistent with that of EF hands 2 and 3 being paired and EF hands 1 and 4 being paired. III - Details of metal coordination in the four EF hands of Hans-LanM. La in EF hands 1, 2, and 3 III The coordination sphere of the ion is composed of the side chain Oδ of N1 (monodentate), the ligands from D3, D5, E9 and E 12 The total coordination number is 10. III -ligand distance is By Shannon, 10 Coordinate La III The crystal radius is given as 6 coordination O 2- The radius is given as La III The -O distance is estimated as This is consistent with our results. The metal ion in EF4 was modeled as Na due to the shorter metal-ligand distance, lower coordination number, and the presence of sodium in the crystallization solution. I . Ca cannot be completely excluded II , as it was present early in the protein purification; however, the protein was treated with Chelex at the end of the purification, and the crystallographic data were consistent with the Na determined by the CheckMyMetal server. I The ion is designated as consistent. The ion is connected through the monodentate D1, N3 and D5 side chains, through the bidentate E 12 The side chain is coordinated through the backbone carbonyl of K113 and through a single solvent molecule (total coordination number 7) in a distorted pentagonal bipyramidal geometry. I -protein ligand distance is The solvent molecules In the case of Mex-LanM, biochemical data and NMR spectroscopy also support that EF4 is a poor lanthanide binding site, and it was modeled as having no metal ion in the NMR solution-state structure.

[0079] Figure 53 Shows Dy III The X-ray crystal structure of the combined Hans-LanM at a resolution of a, One of the dimers in the asymmetric unit, including chains A and B. Note that EF4 is unexpectedly bound by Dy IIIoccupies the position while EF1 is only occupied in chain A. b, The overall structure of the asymmetric unit, which consists of two Hans-LanM dimers. III -Hans-LanM has a different structure, the two dimers (and the monomers within each dimer) are in Dy III -Hans-LanM showed significant differences. EF2-4 was Dy in all chains III The EF1 is occupied and ordered only in chain A; in chains B and C, no metal ions are bound to the EF hand, while in chain D, Dy is bound to the EF hand. III ions but could not model the first five residues (N34-D38) of EF1. III Modeling the decisions in all four EF hands resulted in an anomalous diffraction data set (Tables 9-10, Figures 29-30 ) is supported by biochemical data. III The binding site is weak (see Figure 1 d and Figure 6 ), and based on studies on Mex-LanM, EF2 / 3 is likely to be the tighter binding site. This proposal is supported by the Dy anomaly data (Table 10), and the occupancy of the weak metal binding site may be caused by the high protein concentration used for crystallography. c, Dy III -Details of the metal coordination in the EF hand of Hans-LanM. Three different EF1 structures in the asymmetric unit are shown in the top row. The EF1 metal site is almost identical to the sites in EF2 and EF3 only in chain A (with La III -Hans-LanM, in contrast, has very similar EF1-3 loci. Figure 52 In EF1 (A chain), EF2 and EF3, the coordination ligand is La III -Hans-LanM is the same except that the E9 residues (Glu42, Glu66 and Glu91) have been converted to monodentate coordination, resulting in 9-coordination. III The lower coordination number of Dy is consistent with the lanthanide contraction and is observed with other ligands. III -Ligand distances are mostly La III -Hans-LanM short contract Consistent with this observation, the 9-coordinated Dy III The crystal radius is given as 10-coordinate La III short ( Figure 52 The carboxyl 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 It is a 7-coordinated structure with a pentagonal bipyramidal geometry, and La III -The sodium site in Hans is similar, but the metal-ligand distance is slightly shorter Likewise, these distances are similar to those for the 7-coordinated Dy III consistent with expectations.

[0080] Figure 54 Fluorescence spectra of RE-LanM (Hans-LanM, R100K-Hans-LanM, and Mex-LanM) complexes titrated with citrate as a competitor, monitored by intrinsic protein fluorescence. The fluorescence emission value of the apoprotein was normalized to 1.0. Note that the fluorescence intensity of the Trp residue of Hans-LanM decreases from the RE-bound state to the apo state ( Figure 34 ), while the intensity of the Tyr residue of Mex increases from the RE-bound state to the apo state. Initial conditions: For all experiments, 20 μM protein, 40 μM RE, 20 mM acetic acid, 100 mM KCl, pH 5.0, to which increasing concentrations of citrate were titrated. The citrate concentration at which 50% of each metal was desorbed under these conditions ([citrate] 1 / 2 ) are summarized in Table 11 and plotted in Figure 4 a. a, Hans-LanM; b, R100K-Hans-LanM. La [citrate] in wild type vs. R100K-Hans-LanM 1 / 2 Values ​​and Nd[citrate] 1 / 2 The compression difference between the values ​​indicates that dimerization plays an important role in enhancing the effect of LRE (especially La III ) in the affinity differences of Mex-LanM. c, Mex-LanM. Nd and Dy data have been reported previously. d, Nd [citrate] of each protein 1 / 2 Values ​​and Dy[citrate] 1 / 2 Comparison of the ratios of the values ​​indicated that Hans-LanM had a higher Nd / Dy selectivity than Mex-LanM. By two-tailed t-test, there was no significant difference in the ratios between wild-type Hans-LanM and the R100K variant (p>0.05), indicating that the hydrogen bonding network involving Arg100 was selective for Nd III / HRE selectivity does not contribute much, although it does contribute to La III Selectivity has a significant effect ( Figure 4 a). All data are shown as mean ± sd (ac) or sem (d) of data from three independent experiments.

[0081] Figure 55The separation of a 95:5 Nd:Dy mixture using immobilized Hans-LanM is shown. The desorption protocol consisted of three steps of malonate concentration (30, 50, 90 mM; see right axis) followed by pH 1.5 (HCl). The results reveal that the purity of Dy generated using Hans-LanM is slightly lower than that using the R100K variant (compared to Figure 4 d, with Dy purity of 83.6% and 98%, respectively, with similar yields). Although similar selectivity profiles of the immobilized protein for La to Gd were observed in equilibrium binding experiments with La-Dy, the selectivity patterns diverged at Tb ( Figure 4 c). The selectivity differences between Hans-LanM and R100K variants were confirmed by using a binary Nd / Dy system, as the uncertainty in the determination of the distribution factor for Dy in the 9-element RE group precluded the ability to distinguish small differences in the Dy / Nd separation factors between proteins (Tables 13-14). In this binary Nd / Dy experiment (Table 19), we determined a separation factor of 8.12 ± 0.40 for Hans-LanM and 12.7 ± 1.3 for the R100K variant, which is consistent with the improved Dy separation effect of R100K. Although consistent with the values ​​obtained in the 9-element experiment, this result is slightly different from the equilibrium binding results with free Hans-LanM protein and R100K-Hans-LanM protein, which revealed a similar high selectivity of Nd over Dy ( Figure 4 a, b), which may reflect the weaker LRE-induced dimerization in the R100K variant at low protein concentrations (20 μM) in solution experiments with free protein. The La / Nd selectivity on the column also correlates with the apparent K of free protein (wild type and R100K) in solution. d The values ​​were different, although the experiments with free protein utilized single-element solutions and the effects of mixed metal binding may have affected the on-column data. The R100K variant also performed better on the column, as evidenced by the 2:1 RE:protein stoichiometry. One possible explanation for these results could be that immobilization interferes with dimerization; however, Figure 2 b shows the N- and C-termini of the Hans-LanM dimer, indicating that the C-terminus is approximately This suggests that immobilization itself is not expected to disrupt the interface. However, it must be taken into account that functional dimers would require the two C-termini to be immobilized in close proximity, which is unlikely at the immobilization density of our column. Overall, therefore, we suspect that the dimerization equilibrium is only applicable to a small number of protein units immobilized on the column. We hypothesize that a more thorough exploitation of the dimerization equilibrium in the column format would yield an even more robust separation. The most reliable way to obtain a homogeneous population of dimers on the column would probably be to link the two monomers together (e.g. with a polypeptide chain), thereby modulating the dimerization affinity by mutagenesis of residues contributing to the inter-monomer interaction, and immobilizing the dimer via a single attachment point. Dimerization could also potentially be exploited in other separation formats. These directions are the subject of current work.

[0082] Figure 56 Shown is an SDS-PAGE of S75 gel filtration chromatography from LanM_013, showing high purity. Lanes are labeled with fraction numbers.

[0083] Figure 57 LanM_013 shows approximately 2.0 equivalents of La(III) incorporation in the titration followed by UV-vis spectroscopy. 278 nm data is the top point, 285 nm data is the bottom point. Buffer: 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0, 20 μM LanM_013.

[0084] Figure 58 Shown is the tracking of LanM_013-metal interactions by following protein tyrosine fluorescence. Conditions: 20 mM acetic acid, 100 mM KCl, 5% glycerol, pH 5.0, 20 μM protein.

[0085] Figure 59 Shown is citrate used as a competitor for the removal of bound La(III) from LanM_013 (3 equivalents of La(III) were added before starting the experiment). Conditions: 20 mM acetic acid, 100 mM KCl, 5% glycerol, pH 5.0, 20 μM protein.

[0086] Figure 60 Shown is a xylenol orange competition titration used to directly assess metal binding of LanM_013 at pH 6.0. Conditions: 20 mM MES, 20 mM acetic acid, 100 mM KCl, chelex-treated, pH 6.0, 20 μM protein.

[0087] Figure 61Conformational changes of LanM_013 monitored using circular dichroism spectroscopy are shown. Conditions: (pH 5.0) 20 mM acetic acid, 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.

[0088] Figure 62 Affinity determination of LanM_013 at pH 5.0 is shown. Conditions: 10 μM LanM_013, 20 mM acetic acid, 100 mM KCl, 0-10 mM LaCl3, 10 mM EDTA, pH 5.0 (left). 20 μM LanM_013, 20 mM acetic acid, 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), respectively. The Hill coefficients for both are about 1.5.

[0089] Figure 63 Shown are 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) containing Nd, (B) Hans-LanM (R100K) containing Nd, (C) LanM_012 containing Nd, (D) Mex-LanM (T90W) containing Yb, (E) Hans-LanM (R100K) containing Yb, and (F) LanM_012 containing Yb. Excitation: 280 nm.

[0090] Figure 64 The apparent K of LanM_013 (10 μM) is shown using free lanthanide ion concentrations buffered with EGTA by CD spectroscopy. d Determination of (A)[θ] 222nm Relative to free Gd III A two-phase fit was used to determine the apparent dissociation constant (K d1,app , K d2,app ) and Hill coefficients (n1, n2). (B)[θ] 222nm Relative to free Dy III A single-phase fit was used to determine the apparent dissociation constant (K d,app ) and Hill coefficient (n). (C)[θ] 222nm Relative to free Ho III A single-phase fit was used to determine the apparent dissociation constant (K d,app) and Hill coefficient (n). Buffer: 20 mM acetic acid, 100 mM KCl, 0-10 mM Ln III -EGTA, pH 5.0. Samples were equilibrated for 3 hours before measurement.

[0091] Figure 65 Selectivity curves for column-immobilized lantimedulin variants for the La-Dy REE population are shown. LanM_001 = Mex-LanM. LanM_002 = Hans-LanM. Comparison of distribution values ​​(log D) at total REE equilibrium using a LanM column at pH 5. The plot on the right is identical to the plot on the left, except that it also includes LanM_002 for reference. The calculated separation factors are shown in the table below. Column dimensions: 5 cm x 0.5 cm.

[0092] Figure 66 Selectivity curves for column-immobilized lantimedulin variants for the Gd-Lu and Y REE groups are shown. LanM_001 = Mex-LanM. LanM_002 = Hans-LanM. Comparison of distribution values ​​(log D) at total REE equilibrium using a LanM column at pH 5. The plot on the right is identical to the plot on the left, except that it also includes LanM_002 for reference. The calculated separation factors are shown in the table below. Column dimensions: 5 cm × 0.5 cm.

[0093] Figure 67 Characterization of variant cohort 1 is shown. (A) XO competition assay with titration of Nd(III), pH 6.1. The absorbance of XO at 574 nm is plotted against the Nd(III) equivalents. (B) Conformational changes of LanM variants (20 μM) are monitored by CD at pH 5.0. The ellipticity at 222 nm is plotted against the Nd(III) equivalents.

[0094] Figure 68 Characterization of variant cohort 2 is shown. (A) XO competition assay with titration of Nd(III), pH 6.1. The absorbance of XO at 574 nm is plotted against the Nd(III) equivalents. (B) Conformational changes of LanM variants (20 μM) are monitored by CD at pH 5.0. The ellipticity at 222 nm is plotted against the Nd(III) equivalents.

[0095] Figure 69 Characterization of variant cohort 3 is shown. (A) XO competition assay with titration of Nd(III), pH 6.1. The absorbance of XO at 574 nm is plotted against the Nd(III) equivalents. (B) Conformational changes of LanM variants (20 μM) are monitored by CD at pH 5.0. The ellipticity at 222 nm is plotted against the Nd(III) equivalents.

[0096] Figure 70 Characterization of the "combo" variant group is shown. (A) XO competition assay with titration of Nd(III), pH 6.1. The absorbance of XO at 574 nm is plotted against the Nd(III) equivalent. (B) Conformational changes of LanM variants (20 μM) are monitored by CD at pH 5.0. The ellipticity at 222 nm is plotted against the Nd(III) equivalent.

[0097] Figure 71 Conformational changes of LanM variant cohort 3 (5 μM) monitored by CD at pH 5.0 are shown. Ellipticity at 222 nm is plotted against free Nd(III) concentration. (A) A98G (B) A99G (C) A102G. Buffer: 20 mM acetic acid, 100 mM KCl, 10 mM Nd-EGTA, pH 5.0. DETAILED DESCRIPTION

[0098] Although the claimed subject matter will be described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the advantages and features described herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of this disclosure.

[0099] As used herein, unless otherwise indicated, when used in connection with a measurable variable (such as a parameter, amount, duration, etc.) or a list of alternatives, "about," "substantially," or "similar" is intended to encompass deviations from the specified value, including but not limited to deviations within experimental error, which can be determined, for example, by a given data set, industry-accepted standards, etc., and / or by a given confidence interval (e.g., a 90%, 95% or higher confidence interval for the mean), such as ±10% or less, ±5% or less, ±1% or less, and ±0.1% or less relative to the specified value, as long as such variable deviations and / or alternative deviations are suitable for practicing the present disclosure. As used herein, the term "about" can refer to the amount or value in question being the exact value or the value recited in the claims or taught herein that can produce an equivalent result or effect. That is, it should be understood that amounts, dimensions, compositions, parameters, and other quantities and representations need not and need not be exact, but can be approximate and / or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, etc., or other factors known to those skilled in the art that can produce equivalent results or effects. Generally, whether or not explicitly stated as such, an amount, size, composition, parameter or other quantity or characterization or alternative is "about" or "similar." It is understood that when "about" is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless otherwise expressly stated.

[0100] The range of values ​​disclosed herein. The range specifies a lower limit and an upper limit. Unless otherwise stated, the range includes the lower limit, the upper limit, and all values ​​between the lower limit and the upper limit, including but not limited to all values ​​of the minimum value (lower limit or upper limit) in the range. It should be understood that the use of this range format is for convenience and simplicity, and should therefore be interpreted in a flexible manner to include not only the values ​​explicitly stated as range limits, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a numerical range of "0.1% to 5%" should be interpreted as including not only the values ​​explicitly stated as 0.1% to 5%, but also, unless otherwise stated, individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges within the range (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, as well as other possible subranges). It will also be understood (as described above) that a number of values ​​are disclosed herein, and each value is disclosed herein 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, by using the preposition "about," it can be understood that the particular value forms a further disclosure. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0101] As used herein, unless otherwise indicated, the term "group" refers to a chemical entity that is monovalent (i.e., has one end that can covalently bond to other chemical species), divalent, or polyvalent (i.e., has two or more ends that can covalently bond to other chemical species). The term "group" also includes free radicals (e.g., monovalent and polyvalent, e.g., divalent, trivalent, etc.). Examples of groups include:

[0102]

[0103] Amino acids and amino acid residues may be referred to herein by either their commonly used three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0104] 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, and tryptophan.

[0105] The present disclosure provides proteins that bind rare earth metals. Also provided are devices and kits comprising the proteins of the present disclosure. Also provided are methods of using the proteins and devices.

[0106] 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.

[0107] The Wt Hans-LanM of the present disclosure may be one of the following peptides:

[0108]

[0109]

[0110] The proteins disclosed herein may have various lengths. For example, the proteins disclosed herein have between 65 and 160 amino acid residues, including all integer amino acid values ​​and ranges therebetween. For example, the protein has a molecular weight of approximately 8 kDa to 14 kDa, including all 0.1 Da values ​​and ranges therebetween (e.g., ~12 kDa). The proteins disclosed herein comprise at least one segment that can bind 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) to the sequence of Hansschlegelia quercus LanM (which may be referred to as Hans-LanM). In other various examples, the protein is a truncated form. For example, the protein is truncated at the N-terminus by deleting the first 10, 20, 30, or 40 residues of the complete translated sequence. In another example, the protein is truncated at the C-terminus by deleting the last 10, 20, 30, or 40 residues of the complete translated sequence. In various examples of truncated sequences, EF-hand structures 2 and 3 are retained, as well as most of the hydrophobic core of the protein.

[0111] Suitable Hans-LanM proteins and LanM proteins disclosed herein include wild-type H.quercus LanM proteins, or homologs from other organisms having at least two EF-hand motifs, wherein at least one EF-hand motif has at least 3 carboxylic acid residues and at least two EF-hand motifs are separated by 10-15 residues. References herein are generally to "lantamedulin," "LanM," or "LanM protein," and should be understood to include the wild-type and homologs described herein. "LanM" may include an intact protein having one or more LanM units or a portion thereof comprising one or more LanM units. A LanM unit includes at least two EF-hand motifs, wherein at least one EF-hand motif has at least 3 carboxylic acid residues and at least two EF-hand motifs are separated by 10-15 residues. For ease of reference, reference will be made to lantamedulin, LanM, or LanM protein in the discussion and should be understood to include both intact proteins and portions of intact proteins having suitable LanM units.

[0112] Various substitutions can be made to the polypeptides or proteins of the present disclosure. For example, one or more amino acid residues can be substituted with different amino acid residues. The amino acid residues can be standard or non-standard amino acid residues. For example, R100 can be substituted with an amino acid residue. For example, the arginine can 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) to any of the aforementioned variants. The residue numbering corresponds to the residue numbering when a 23-residue signal peptide is present. For proteins lacking a signal peptide, the first residue after cleavage of the N-terminal methionine is residue 24.

[0113] In various embodiments, the protein of the present disclosure has a residue suitable for fixation to a matrix. The residue can be part of a large sequence consisting of 2 to 10 amino acid residues. For example, the residue comprises a functional group that chemically reacts with other functional groups on the matrix, thereby covalently attaching the residue (and thus the protein) to the matrix. For example, the matrix can comprise a maleimide group that reacts with nucleophilic groups (such as the thiol group of cysteine ​​or the amine group of lysine). Other suitable chemical methods (such as click chemistry) are known in the art and can be used. For example, the matrix can be a resin or bead comprising a functional group that reacts with the residues of the LanM protein. For example, the functional group can be maleimide, alkyne or azide.

[0114] In various examples, the protein or peptide capable of binding to a metal / metal ion of the present disclosure (e.g., the protein or peptide capable of dimerizing upon contact with a metal / metal ion of the present disclosure) may comprise 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 (e.g., 12 amino acid residues). Each EF-hand motif is separated by 12 or 13 amino acid residues, wherein each amino acid residue is any standard 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, wherein each X is any standard 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 -X5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E. For the first EF-hand motif (ie, EF1), the second EF-hand motif (ie, 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 Each independently represents any standard amino acid residue. In various examples, X of EF1, EF2 and / or EF4 7 Is T or S. For EF3, X 1 It is N; X 3 It is D; X 4 It is G or A; X 5 Is D or N; X 7 Is T or S; X 8 is a hydrophobic residue, X 9 It is E; X 10 is a hydrophobic residue; X 11 is D; and X 2 and X 6 Each is independently any standard 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, EF3's X 8 is L. In various examples, EF3's X 10 is L, I, or M. Without intending to be bound by any particular theory, it is believed that the dimerization strength of this protein 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.

[0115] In various examples, the proteins or peptides disclosed herein may have enhanced REE / REE selectivity. This selectivity may be between light rare earth metals and heavy rare earth metals and exceed the selectivity of M. extorquens lanthanum. Such a protein may comprise 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, and each EF-hand motif is separated by 12 or 13 amino acid residues, wherein each residue is a standard 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:

[0116] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E.

[0117] For the first EF-hand motif (ie, EF1) and the fourth EF-hand motif (ie, 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 Each independently represents any standard amino acid residue. In various examples, X of EF1 and / or EF4 7 Is T or S. For EF2, X 1 It is N; X 3 It is D; X 5 It is D; X 7 Is T or S; X 8 is a hydrophobic residue; X 9 It is E; X 12 is E; and X 2 、X 4 、X 6 、X 10 and X 11 Each is independently any standard 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, IM or V. For EF3, X 1 It is D; X 3 It is D; X 5 It is D; X 6 It 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 Each independently is any 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 of EF2 or EF3 2 is P, and a protein having this sequence can be linked to another protein of the present disclosure via a peptide linker described herein.

[0118] In various embodiments, the second EF hand has the following sequence:

[0119] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E,

[0120] in

[0121] X 1 is D or N;

[0122] X 2 is any standard amino acid;

[0123] X 3 is D, N or E;

[0124] X 4is any standard amino acid;

[0125] X 5 is D, N or E;

[0126] X 6 is any standard amino acid;

[0127] X 7 is any standard amino acid;

[0128] X 8 is a hydrophobic residue;

[0129] X 9 independently D, E, or T;

[0130] X 10 is a hydrophobic residue; and

[0131] X 11 Any standard amino acid.

[0132] In various other embodiments, the second EF hand has the following sequence:

[0133] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E,

[0134] in

[0135] X 1 It is N;

[0136] X 2 is any standard amino acid;

[0137] X 3 It is D;

[0138] X 4 is any standard amino acid;

[0139] X 5 It is D;

[0140] X 6 is any standard amino acid;

[0141] X 7 It is T or S;

[0142] X 8 is a hydrophobic residue;

[0143] X 9 It is E;

[0144] X 10 is any standard amino acid; and

[0145] X 11 Any standard amino acid.

[0146] In various embodiments, the second EF hand's X 2 is P; in various embodiments, X 7 is T or S; in various embodiments, X 8 It is L, I, M or V.

[0147] In various embodiments, the third EF hand has the following sequence:

[0148] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E,

[0149] in

[0150] X 1 It is N;

[0151] X 2 is any standard amino acid;

[0152] X 3 It is D;

[0153] X 4 It is G or A;

[0154] X 5 is D or N;

[0155] X 6 is any standard amino acid;

[0156] X 7 It is T or S;

[0157] X 8 is a hydrophobic residue;

[0158] X 9 It is E;

[0159] X 10 is a hydrophobic residue; and

[0160] X11 It’s D.

[0161] In various other embodiments, the third EF hand has the following sequence:

[0162] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E,

[0163] in

[0164] X 1 It is D;

[0165] X 2 is any standard amino acid;

[0166] X 3 is D;

[0167] X 4 is D;

[0168] X 5 is D;

[0169] X 6 is G;

[0170] X 7 It is T or S;

[0171] X 8 is a hydrophobic residue;

[0172] X 9 It is D;

[0173] X 10 is any standard amino acid; and

[0174] X 11 Any standard amino acid.

[0175] 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 It is L, I or M.

[0176] The proteins of the present disclosure may comprise 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).

[0177] The proteins of the present disclosure may comprise 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).

[0178] 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).

[0179] The proteins described in the present disclosure can 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).

[0180] Examples of proteins disclosed herein include, but are not limited to, the following:

[0181] >Hans-LanM (complete protein sequence, signal peptide is underlined)

[0182]

[0183] >Hans-LanM (as expressed herein, signal peptide removed)

[0184]

[0185] >Hans-LanM(R100K) (as expressed in this study, signal peptide removed, substitutions are underlined)

[0186]

[0187] >Hans-LanM-Cys (for immobilization)

[0188]

[0189] >Hans-LanM(R100K)-Cys (for immobilization)

[0190]

[0191] >LanM_012 (Xanthobacter flavus, Flavobacterium, signal peptide removed)

[0192]

[0193] >LanM_013 (expressed construct; signal peptide removed, Met added to the N-terminus, EF hand underlined):

[0194]

[0195] LanM_013 (full-length sequence of Methyloligella sp. GL2 with signal peptide underlined):

[0196]

[0197] >Methyloligella halotolerans (expected to behave similarly to LanM_013; sequence with signal peptide removed)

[0198]

[0199] >Mex-LanM-A32D / A117K (mutations are underlined)

[0200]

[0201] >Mex-LanM-A32D / A117R (mutations are underlined)

[0202]

[0203] >Mex-LanM-I42L / N108D / I115L (mutations are underlined)

[0204]

[0205] >LanM_011 (Hyphomicrobium, signal peptide removed)

[0206]

[0207] >Unclassified Hyphomicrobium (signal peptide removed; shown to have only 3 functional EF hands)

[0208]

[0209] >HansR100K-L1

[0210]

[0211]

[0212] >HansR100K-L2

[0213]

[0214] >HansR100K-L3

[0215]

[0216] >HansR100K-L4

[0217]

[0218] >HansR100K-L5

[0219]

[0220] >Mex-LanM-G51A (mutation is underlined)

[0221]

[0222] >Mex-LanM-A98G (mutation is underlined)

[0223]

[0224] >Mex-LanM-A99G (mutation is underlined)

[0225]

[0226] >Mex-LanM-V100G (mutations are underlined)

[0227]

[0228] >Mex-LanM-A102G (mutation is underlined)

[0229]

[0230] >Hans-LanM-I43A (mutation is underlined)

[0231]

[0232] >Hans-LanM-I43V (mutation is underlined)

[0233]

[0234] >Hans-LanM-A44N (mutation is underlined)

[0235]

[0236] >Hans-LanM-A44S (mutation is underlined)

[0237]

[0238] >Hans-LanM-A44T (mutation is underlined)

[0239]

[0240] >Hans-LanM-I47A (mutation is underlined)

[0241]

[0242] >Hans-LanM-I47V (mutation is underlined)

[0243]

[0244] >Hans-LanM-M92L (mutation is underlined)

[0245]

[0246] >Hans-LanM-M92A (mutation is underlined)

[0247]

[0248] >Hans-LanM-M92D (mutations are underlined)

[0249]

[0250] >Hans-LanM-D93A (mutation is underlined)

[0251]

[0252] >Hans-LanM-D93N (mutation is underlined)

[0253]

[0254] >Hans-LanM(3E9Q)

[0255]

[0256] >Mex-LanM-N108D / A124G (mutations are underlined)

[0257]

[0258] >Mex-LanM-N108D / A127G (mutations are underlined)

[0259]

[0260] >Mex-LanM-N108D / A102G (mutations are underlined)

[0261]

[0262] >Mex-LanM-N108D

[0263]

[0264] Without intending to be bound by any particular theory, it is believed that at least a portion of the LanM protein disclosed herein dimerizes with additional LanM monomers upon contact with a rare earth metal. The LanM monomers in the dimer can be identical or different. It is believed that at least the following sequence dimerizes:

[0265]

[0266]

[0267]

[0268] Without wishing to be bound by any particular theory, it is believed that the LanM sequence can dimerize with or without a signal peptide included in the sequence.

[0269] In various examples, two proteins of the present disclosure can be covalently conjugated together. For example, the two proteins can be conjugated via a peptide linker. For example, the linker can be (GGS) nmotif, wherein n is 2, 3, 4, 5 or 6. For example, the linker can be GGSGGSGGSGGSGGSGGS (SEQ ID NO: 43). Examples of conjugated proteins include, but are not limited to:

[0270] >HansR100K-L1

[0271]

[0272] >HansR100K-L2

[0273]

[0274] >HansR100K-L3

[0275]

[0276] >HansR100K-L4

[0277]

[0278] >HansR100K-L5

[0279]

[0280]

[0281] The above 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.

[0282] Without wishing to be bound by any particular theory, it is believed that LanM_013, methyloligella halotolerans and LanM_011 do not form dimers.

[0283] In aspects of the present disclosure, a device is provided. The device comprises one or more proteins of the present disclosure.

[0284] The proteins described herein can be included in various devices. Non-limiting examples of devices include filters, membranes, sensors, handheld detectors, microplate readers, fluorometers, biosensors, online monitors, and the like.

[0285] In aspects of the present disclosure, kits are provided. The kits may provide one or more proteins of the present disclosure and / or one or more devices of the present disclosure. The kits may include instructions for use of the proteins or devices.

[0286] Various methods of using the proteins and / or devices of the present disclosure are provided in aspects of the present disclosure. The methods of the present disclosure can be used to bind one or more lanthanides and / or actinides, or to detect and / or quantify the amount of one or more lanthanides and / or actinides.

[0287] Methods using proteins and / or devices of the present disclosure can be methods of binding one or more rare earth metals (e.g., lanthanides and / or actinides) in a sample. The 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 of contaminated soil, drainage water (e.g., mine drainage water, such as acid mine drainage water), or leachate (e.g., electronic waste leachate or ore leachate). In various other examples, the sample is a solid sample. The method can be applied to samples of 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.

[0288] Various lanthanides (e.g., lanthanide ions) and / or actinides (e.g., actinide ions) can be bound by proteins and / or devices. Examples of lanthanides and actinides that can 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 can be 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 can be the same or different. The concentration of lanthanides and / or actinides in the sample can 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).

[0289] In various examples, one or more lanthanides and / or actinides bound to one or more proteins and / or devices can be separated and recovered from the proteins and / or devices. The lanthanides and / or actinides can be unbound 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, if 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 dissociated from the protein. As an illustrative example, if both Nd and Dy are bound, one metal can be selectively dissociated while the other metal remains bound. For example, one metal can be dissociated by contact with a chelating agent, while the other metal can be dissociated by adjusting the pH. After the one or more lanthanides are unbound and separated, the one or more proteins and / or devices can be reused.

[0290] The method of the present disclosure can be a method for detecting and / or quantifying the amount of one or more lanthanides and / or actinides in a sample. The method can include contacting the sample with one or more proteins and / or devices of the present disclosure. The contacted sample can then be exposed to light, and the exposed contacted sample thereby produces emitted light. The resulting emitted light result can then be compared with a known standard curve for a specific lanthanide or actinide. The concentration can be determined by this comparison. A known standard curve can be prepared based on the desire to detect and / or determine the amount of any specific lanthanide or actinide. Methods for preparing standard curves are known in the art.

[0291] The method of detecting and / or quantifying can be performed on a variety of samples. Non-limiting examples of samples include drinking water, wastewater, groundwater, ash ponds, aqueous extracts of contaminated soil, drainage water (such as mine drainage water, such as acid mine drainage) or leachate (such as electronic waste leachate or ore leachate). In various other examples, the sample is a solid sample. The method can be applied to samples of various pH values. For example, the sample has a pH value of 6 or lower, for example, 5.5 or lower, 5 or lower, 4.5 or lower, 4 or lower, 3.5 or lower, 3 or lower. In various examples, the pH value is greater than 6.

[0292] Various lanthanides (e.g., lanthanide ions) and / or actinides (e.g., actinide ions) can 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 ions thereof. The bound lanthanides and / or actinides can be the same or different. The concentration of the lanthanides and / or actinides in the sample can be less than 1 ppm.

[0293] The following statements provide various examples and embodiments of the present disclosure.

[0294] Statement 1. A protein capable of binding a metal and / or metal ion (e.g., in various examples, the protein is capable of dimerizing 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 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 have 12 amino acid residues, each EF-hand motif has the following sequence:

[0295] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E,

[0296] in

[0297] i) For the first EF-hand motif, the second EF-hand motif, and the fourth EF-hand motif:

[0298] Each X 1 independently D or N;

[0299] Each X 2 independently any standard amino acid;

[0300] Each X 3 independently D, N, or E;

[0301] Each X 4 independently any standard amino acid;

[0302] Each X 5 independently D, N, or E;

[0303] Each X 6 independently any standard amino acid;

[0304] Each X 7 independently any standard amino acid;

[0305] Each X 8 are independently hydrophobic residues;

[0306] Each X 9 independently D, E, or T;

[0307] Each X 10are independently hydrophobic residues; and

[0308] Each X 11 independently any standard amino acid;

[0309] ii) For the third EF-hand motif:

[0310] X 1 It is N;

[0311] X 2 is any standard amino acid;

[0312] X 3 It is D;

[0313] X 4 It is G or A;

[0314] X 5 is D or N;

[0315] X 6 is any standard amino acid;

[0316] X 7 It is T or S;

[0317] X 8 is a hydrophobic residue;

[0318] X 9 It is E;

[0319] X 10 is a hydrophobic residue; and

[0320] X 11 is D; and

[0321] iii) The EF-hand motifs are connected by a 12 or 13 amino acid residue linker, and each amino acid residue of the linker is a standard amino acid, except for the third EF-hand motif and the fourth EF-hand motif, which are connected by the following sequence: (X)5-R-(X)6, wherein each X is independently a standard amino acid, and at least one amino acid of any linker is hydrophobic.

[0322] Statement 2. The protein according to Statement 1, wherein the X of the first EF-hand motif, the second EF-hand motif and / or the fourth EF-hand motif 7 are independently T or S.

[0323] Statement 3. A protein according to Statement 1 or Statement 2, wherein the X of the third EF-hand motif 4 It’s A.

[0324] Statement 4. A protein according to any one of the preceding statements, wherein X of the third EF-hand motif8 It’s L.

[0325] Statement 5. A protein according to any one of the preceding statements, wherein X of the third EF-hand motif 10 It is L, I or M.

[0326] Statement 6. A protein with enhanced REE / REE 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 have 12 amino acid residues, each EF-hand motif has the following sequence:

[0327] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -E,

[0328] in

[0329] i) For the first EF-hand motif and the fourth EF-hand motif:

[0330] Each X 1 are independently D or N;

[0331] Each X 2 independently any standard amino acid;

[0332] Each X 3 independently D, N, or E;

[0333] Each X 4 independently any standard amino acid;

[0334] Each X 5 are independently D, N or E;

[0335] Each X 6 independently any standard amino acid;

[0336] Each X 7 independently any standard amino acid;

[0337] Each X 8 are independently hydrophobic residues;

[0338] Each X 9independently D, E, or T;

[0339] Each X 10 are independently hydrophobic residues; and

[0340] Each X 11 independently any standard amino acid;

[0341] ii) For the second EF-hand motif:

[0342] X 1 It is N;

[0343] X 2 is any standard amino acid;

[0344] X 3 It is D;

[0345] X 4 is any standard amino acid;

[0346] X 5 It is D;

[0347] X 6 is any standard amino acid;

[0348] X 7 It is T or S;

[0349] X 8 is a hydrophobic residue;

[0350] X 9 It is E;

[0351] X 10 is any standard amino acid; and

[0352] X 11 is any standard amino acid; and

[0353] iii) For the third EF-hand motif:

[0354] X 1 It is D;

[0355] X 2 is any standard amino acid;

[0356] X 3 is D;

[0357] X 4 is D;

[0358] X 5 is D;

[0359] X 6 is G;

[0360] X7 It is T or S;

[0361] X 8 is a hydrophobic residue;

[0362] X 9 It is D;

[0363] X 10 is any standard amino acid; and

[0364] X 11 is any standard amino acid;

[0365] iv) at least one X in the second EF-hand motif and the third EF-hand motif 2 is P; and

[0366] v) The EF-hand motifs are connected by a 12 or 13 amino acid residue linker, wherein each amino acid of the linker is a standard amino acid and at least one amino acid of any linker is hydrophobic.

[0367] Statement 7. The protein according to Statement 6, wherein the X of the second EF-hand motif and / or the third EF-hand motif 8 It is L, I, M or V.

[0368] Statement 8. The protein of any of the preceding statements, wherein the protein comprises the following sequence: MKLSLKAGAA ITAFVFAASP VLAASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGETKGRLTEKDWA RANKDGDQTL EMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 1);

[0369] or

[0370] MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWA

[0371] RANKDGDQTL EMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK(SEQ ID NO:2);

[0372] or a protein having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2, or a protein comprising the following sequence: MLTGKEFLRKYNKDKDSTVEIVEAIDLGTKVFKAINPDKD K TLEAAETKGRLSDEDWAQFNKDGDKTLELDEWLIIVRKRFNDADANKDGKLTEAELDAPAGQQLILLIAK(SEQ ID NO:7);

[0373] or a protein with 70% identity thereto.

[0374] Statement 9. The protein of any of the preceding statements, wherein said protein is complexed with a rare earth element.

[0375] Statement 10. The protein of Statement 9, wherein said rare earth element is a light rare earth element.

[0376] Statement 11. The protein of Statement 9, wherein said rare earth element is a heavy rare earth element.

[0377] Statement 12. The protein of any of the preceding statements, wherein said protein comprises the sequence:

[0378]

[0379] wherein X is any standard amino acid residue except R.

[0380] Statement 13. The protein of Statement 12, wherein said protein comprises the sequence:

[0381]

[0382] Statement 14. The protein of any one of Statements 1-12, wherein the protein has the following sequence: >Hans-LanM (complete protein sequence, signal peptide is underlined)

[0383]

[0384] >Hans-LanM (as expressed herein, signal peptide removed)

[0385]

[0386] >Hans-LanM(R100K) (signal peptide removed, substitutions are underlined)

[0387]

[0388] >Hans-LanM-Cys (for immobilization)

[0389]

[0390] >Hans-LanM(R100K)-Cys (for immobilization)

[0391]

[0392] >LanM_012 (Flavobacterium, signal peptide removed)

[0393]

[0394] >LanM_013 (expressed construct; signal peptide removed, Met added to the N-terminus, EF hand underlined):

[0395]

[0396] LanM_013 (full-length sequence of Methyloligella sp. GL2 with signal peptide underlined):

[0397]

[0398] >Methyloligella halotolerans (expected to behave similarly to LanM_013; sequence with signal peptide removed)

[0399]

[0400] >Mex-LanM-A32D / A117K (mutations are underlined)

[0401]

[0402] >Mex-LanM-A32D / A117R (mutations are underlined)

[0403]

[0404] >Mex-LanM-I42L / N108D / I115L (mutations are underlined)

[0405]

[0406] >LanM_011 (Hyphomicrobium, signal peptide removed)

[0407]

[0408] >Unclassified Hyphomicrobium (signal peptide removed; shown to have only 3 functional EF hands)

[0409]

[0410] >HansR100K-L1

[0411]

[0412] >HansR100K-L2

[0413]

[0414] >HansR100K-L3

[0415]

[0416] >HansR100K-L4

[0417]

[0418] >HansR100K-L5

[0419]

[0420]

[0421] >Mex-LanM-G51A (mutation is underlined)

[0422]

[0423] >Mex-LanM-A98G (mutation is underlined)

[0424]

[0425] >Mex-LanM-A99G (mutation is underlined)

[0426]

[0427] >Mex-LanM-V100G (mutations are underlined)

[0428]

[0429] >Mex-LanM-A102G (mutation is underlined)

[0430]

[0431] >Hans-LanM-I43A (mutation is underlined)

[0432]

[0433] >Hans-LanM-I43V (mutation is underlined)

[0434]

[0435] >Hans-LanM-A44N (mutation is underlined)

[0436]

[0437] >Hans-LanM-A44S (mutation is underlined)

[0438]

[0439] >Hans-LanM-A44T (mutation is underlined)

[0440]

[0441] >Hans-LanM-I47A (mutation is underlined)

[0442]

[0443] >Hans-LanM-I47V (mutation is underlined)

[0444]

[0445] >Hans-LanM-M92L (mutation is underlined)

[0446]

[0447] >Hans-LanM-M92A (mutation is underlined)

[0448]

[0449] >Hans-LanM-M92D (mutations are underlined)

[0450]

[0451] >Hans-LanM-D93A (mutation is underlined)

[0452]

[0453] >Hans-LanM-D93N (mutation is underlined)

[0454]

[0455] >HansR100K-L1

[0456]

[0457] >HansR100K-L2

[0458]

[0459] >HansR100K-L3

[0460]

[0461] >HansR100K-L4

[0462]

[0463] >HansR100K-L5

[0464]

[0465] >Hans-LanM(3E9Q)

[0466]

[0467] >Mex-LanM-N108D / A124G (mutations are underlined)

[0468]

[0469] >Mex-LanM-N108D / A127G (mutations are underlined)

[0470]

[0471] >Mex-LanM-N108D / A102G (mutations are underlined)

[0472]

[0473] >Mex-LanM-N108D

[0474]

[0475] Statement 15. The protein of any one of Statements 1-12, wherein said protein has the sequence:

[0476]

[0477]

[0478] Statement 16. A device comprising a protein according to any preceding statement.

[0479] Statement 17. The device of Statement 16, wherein said device is a filter, a membrane, a sensor, a handheld detector, a microplate reader, a fluorometer, a biosensor, or an online monitor.

[0480] 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.

[0481] Statement 19. A method of separating rare earth elements, comprising contacting a protein according to any one of Statements 1 to 15 with a sample containing a rare earth element, wherein the rare earth element binds to one or more proteins according to any one of Statements 1 to 15, and removing the proteins from the sample.

[0482] Statement 20. The method of statement 19, wherein the sample is drinking water, wastewater, groundwater, ash pond, aqueous extract from contaminated soil, drainage water, leachate, solid waste (e.g., electronic waste), or aqueous extract or leachate of ore or tailings, or a solid sample.

[0483] Statement 21. A method according to Statement 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.

[0484] Statement 22. The method of any one of Statements 19 to 21, wherein the method further comprises detecting and quantifying the one or more rare earth elements.

[0485] Statement 23. The method of any one of Statements 19 to 22, wherein a plurality of different rare earth elements are bound to the protein.

[0486] Statement 24. The method of Statement 23, wherein each different rare earth element is individually separated from the protein.

[0487] Statement 25. A method of determining the presence of a light rare earth element in a sample, comprising contacting the sample with a protein according to any one of Statements 1 to 15, and determining whether the protein forms a dimer.

[0488] The following examples provide various examples of the present disclosure. They are not intended to be limiting in any form.

[0489] Example 1

[0490] This example describes the use of the peptides / proteins of the present disclosure.

[0491] Technologically critical rare earth elements (REEs) are notoriously difficult to separate due to subtle differences in ionic radius and coordination number between them. The natural lanthanide-binding protein lanthanomodulin offers a sustainable alternative to conventional solvent extraction-based separations. Here, we characterize a novel lanthanomodulin from Hansschlegelia quercus (Hans-LanM) with an oligomeric state sensitive to REE ionic radius, with lanthanum(III)-induced dimers >100-fold more compact than those induced by dysprosium(III). X-ray crystal structures reveal how the picometer-scale difference in radius between lanthanum(III) and dysprosium(III) is propagated into the quaternary structure of Hans-LanM via a carboxylate shift that rearranges the second-sphere hydrogen-bonding network. Comparison with the prototypical lanthanomodulin from Methylorubrum extorquens reveals a different metal coordination strategy, rationalizing the enhanced selectivity of Hans-LanM for REEs. Finally, structure-induced mutagenesis of key residues at the Hans-LanM dimer interface modulated dimerization in solution and enabled single-stage, column-based separation of a neodymium(III) / dysprosium(III) mixture to >98% individual elemental purity. This disclosure demonstrates the natural diversity of selective lanthanide recognition motifs and reveals how rare-earth-sensitive dimers can modulate the performance of biomolecule-based separation processes through biological principles.

[0492] This article describes lantamedulin (Hans-LanM) from Hansschlegelia quercus, a methylotrophic bacterium isolated from English oak buds. Hans-LanM exhibits enhanced RE separation ability compared to Mex-LanM. While Mex-LanM is always monomeric, Hans-LanM exists in a monomer / dimer equilibrium, the position of which depends on the specific RE bound. Three X-ray crystal structures and structure-induced mutagenesis of lantamedulin explain the RE-dependent oligomeric state of Hans-LanM and its enhanced separation ability compared to Mex-LanM. Furthermore, a single-stage separation of the important neodymium / dysprosium pair based on Hans-LanM is described. These results illustrate how molecular interactions, common in proteins but rare in small molecules, can be exploited to improve RE separations.

[0493] Unique selectivity profile of Hans-LanM. Several features of lanmodulin have been proposed. First, the LanM protein possesses four EF-hand motifs. The EF-hands consist of a 12-residue flanked by an α-helical carboxylic acid-rich metal-binding loop that is generally responsive to Ca. II However, in Mex-LanM, EF-hands 1-3 bind with low picomolar affinity and superior to Ca II of 10 8 fold selectivity towards lanthanide(III) ions, leading to a large lanthanide-selective disordered-to-ordered conformational transition. EF4 binds with only micromolar affinity. Second, adjacent EF hands in LanM are separated by 12-13 residues, rather than Ca II The typical 25 residues in responsive EF-hand proteins form an unusual three-helix bundle architecture with metal binding sites at the periphery. Third, at least one EF-hand contains a proline at position 2 (in Mex-LanM, all four EF-hands have a P2 residue). Using the first two criteria and a sequence length of less than 200 residues to search the database, 696 putative LanMs were identified. These sequences were visualized using a sequence similarity network to identify LanM sequences that clustered independently of Mex-LanM. At a 65% identity threshold, a subgroup of 642 sequences was formed that was distant from the main group ( Figure 1 a). This exclusive group ("Hans group") includes bacteria from several genera, including Hansschlegelia and Xanthobacter ( Figure 50 ), all of which are facultative methylotrophic bacteria.

[0494] Hans-LanM is characterized by a low sequence identity (33%) with Mex-LanM ( Figure 5 ) and different EF-hand motifs, especially at the first, second, and ninth positions ( Figure 1 b) These positions are important in Mex-LanM and other EF-hand proteins. Therefore, Hans-LanM provides an opportunity to identify features essential for lanthanide-selective recognition by lanthanomodulin.

[0495] Hans-LanM is expressed in E. coli as a 110 amino acid protein ( Figure 5 ). Select La III and Nd III As a representative LRE and select Dy III As a representative HRE for complex studies, this protein bound approximately 3 equivalents of La, similar to Mex-LanM, by ICP-MS. III and Nd III, and combined with a slightly smaller amount of Dy III (Table 1). Also like Mex-LanM, Hans-LanM exhibits very low helicity in the absence of metal, as judged by the circular dichroism (CD) signal at 222 nm ( Figure 1 c) Unexpectedly, only 2 equivalents of La III or Dy III is sufficient to induce a complete conformational change in Hans-LanM ( Figure 6 ), which indicates that the third binding equivalent is weak and does not increase the helicity.

[0496] The apparent dissociation constant (K) determined by CD spectroscopy d,app ) reflects the RE / RE and RE / non-RE selectivity of Mex-LanM under competitive RE recovery conditions. Therefore, similar K values ​​were obtained for Hans-LanM with free metal concentrations controlled by competitive chelating agents. d,app Determine; Result( Figure 1 d, Table 2) is different from Mex-LanM. III and Nd III Binding to Hans-LanM increased the molar ellipticity at 222 nm by a factor of 2.3, which is clearly a complete conformational change in stoichiometric titration. This conformational change is cooperative (Hill coefficient n is 2, Table 2), and K d,app The values ​​are similar, 68 and 91 pM, respectively. In contrast, although Dy III The overall response induced by La III same( Figure 6 ), but in the chelating agent buffered Dy III In the titration, the conformational response of Hans-LanM was smaller (increased by 1.8 times). This difference indicates that Dy III At least one of the binding sites is very weakly responsive (K d,app >0.3 μM, the highest concentration achievable in titrations with chelating buffer). III The main response occurred at 2.6 nM, which was more than 30-fold higher than that with LRE, and had little or no synergy (n=1.3). In contrast, Mex-LanM showed only a general preference for LRE (about 5-fold, Figure 1 e) and all lanthanides and Y III Hans-LanM showed a weak response to calcium (II) (K d,app =60μM), and also for Dy III Lack of cooperativity (n = 1.0) and obvious partial conformational changes ( Figure 51 Thus, Hans-LanM exhibits stronger discrimination between the LRE and HRE than Mex-LanM, with the HRE complex exhibiting lower affinity, weaker cooperativity, and smaller primary conformational changes.

[0497] LRE selective dimerization. The different behaviors of LRE and HRE-Hans-LanM complexes indicate that Mex-LanM does not have an LRE / HRE selective mechanism. Since Mex-LanM complexes similarly with LRE and HRE in monomeric form, it is believed that LRE and HRE can induce different oligomeric states in Hans-LanM. III In the presence of , the molecular weight (MW) of Hans-LanM eluted from the size exclusion chromatography (SEC) column was not the expected 11.9 kDa, but 27.8 kDa, indicating that the dimer ( Figure 7 , Figure 8 a). Apparent molecular weight from Nd III Then it gradually began to decline and III There is a sharp drop towards the expected apparent molecular weight for the monomer ( Figure 2 a. Figure 8 , Table 3). It is worth noting that it is heavier than Gd III The lanthanides do not seem to support the growth of RE-utilizing bacteria.

[0498] To further support preferential dimerization in the presence of physiologically relevant LREs, multi-angle light scattering (MALS) was used to analyze the RE complex of Hans-LanM ( Figure 2 a, Figure 9 ). La III 、Nd III and Gd III The complex has a molecular weight of 22-25 kDa, which is indicative of a dimer, but from Tb III Initially, the molecular weight decreases until Dy III and Ho III , with a molecular weight of approximately 15 kDa (Table 17), which is consistent with the SEC data. II The molecular weight of Hans-LanM is also 14.7 kDa. II The complex and the apo-Hans-LanM complex are still one-third larger than expected for the monomer, indicating that under these conditions, these forms exist in a rapid equilibrium with a monomer:dimer ratio of approximately 2:1. The binding of apo, LanM and Hans to the complex was determined by isothermal titration calorimetry (ITC). III and combined with DyIII Hans-LanM dimerization K d (K 二聚体 )( Figure 10-12 , Table 18). Apoprotein and Dy III The binding proteins dimerize weakly with K values ​​of 117 μM and 60 μM, respectively. 二聚体 , which is consistent with the ratio of monomers and dimers reflected in SEC and MALS traces. III In the presence of , the dimer is too compact to allow monomerization to be observed by ITC, indicating that K 二聚体 <0.4μM( Figure 12 ). Therefore, La III favors dimerization of Hans-LanM over Dy III >100 times.

[0499] with La III Composite Hans-LanM High-resolution X-ray crystal structure demonstrates LRE-induced dimerization ( Figure 52 , Table 4). Two LanM monomers interact head-to-tail ( Figure 2 b), buried by hydrophobic and polar contacts. The surface area ( Figure 2 cd). These interactions occur primarily between side chains contributed by core helices α2 (between EF1 / EF2) and α3 (between EF3 / EF4). Figure 13 The residues at the dimer interface directly contact only one of the four metal binding sites, EF3; the three residues of EF3 in each monomer form a hydrogen bonding network with Arg100 of other monomers ( Figure 2 c), indicating that the occupancy and coordination geometry of this site can control the oligomeric state.

[0500] Small angle X-ray scattering (SAXS) was also used to characterize the relationship between Hans-LanM and its reaction with 3 equivalents of La. III 、Nd III and Dy III The complexes were analyzed ( Figure 14-15 The solvent envelope calculated from SAXS data is similar to La III -Hans-LanM crystallized Hans-LanM dimer fits well with Nd III -Hans-LanM fits well, but not with Dy III -The Hans-LanM fit is poor ( Figure 2 e, Figure 16-18 ). DyIII The weak dimerization of -Hans-LanM is also supported by quantitative indicators such as Porod volume (Table 5-6, Figure 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.

[0501] Structural basis of dimerization. III The structure of -Hans-LanM additionally provides the first detailed view of the coordination environment of lanmodulin and indeed of any natural biomolecule responsible for reversible lanthanide recognition. Previous NMR structures of Mex-LanM revealed the unusual fold of the protein but could not provide molecular details of the metal binding site. To understand the basis for the LRE / HRE distinction, the Dy III -Hans-LanM Finally, this paper reports the Nd III -Mex-LanM resolution structure, which reasonably explains the weaker RE selectivity trend of Mex-LanM.

[0502] In La III -Hans-LanM, EF1-3 by La III Ion occupancy ( Figure 52 be). EF4 is structurally unique and does not exhibit the same III Consistent anomalous difference density and with Na I Modeling was performed ( Figure 21 a) As observed in the lanthanide-dependent methanol dehydrogenase structure, each La III The binding sites are all deca-coordinated ( Figure 22 ). Monodentate Asn (N1 position), four bidentate Glu / Asp residues (D3, D5, E9 and E 12 ) and backbone carbonyl groups (T7 / S7) complete the first coordination sphere in EF1-3 ( Figure 3 a). No exogenous solvent ligands were observed ( Figure 21 b); for Eu III -Hans-LanM performed luminescence studies to determine the number of coordinated solvent molecules (q), yielding q = 0.11, consistent with the X-ray structure of the solvent-free ligand ( Figure 23 ).

[0503] The lanthanide binding sites in Hans-LanM additionally share extensive second-sphere interactions that can further constrain the position of the ligand and the size of the metal-binding pore ( Figure 24This phenomenon is most evident in EF3, where the dimer interface mediates an extended hydrogen bonding network involving several ligands. Arg100, contributed by the adjacent monomer, is projected to the solvent-exposed side of EF3 to contact two carboxylate ligands, Asp85 (D3) and Glu91 (E9), which strengthens their dual-ligand binding mode. Arg100 is also supported by Asp93 (EF3 D 11 ), which is unique to EF3 in Hans-LanM and not observed in Mex-LanM. The importance of this network in Hans-LanM dimerization was tested by making a minimal mutation, R100K. R100K-Hans-LanM has a K of almost the same as wild-type Hans-LanM. d,app Value and Nd III and Dy III response, but for La III K d,app The value is 2 times weaker (Table 7, Figure 25 ). SEC-MALS analysis showed that apo-, La III - and Dy III -R100K-Hans-LanM has a molecular weight of 10-13 kDa ( Figure 26 , Table 8), which indicates an increase in monomerization (especially for La III complex), and suggests that weaker dimerization may be responsible for La III The reason for the lower affinity. All four residues including the Arg100-EF3 network are completely conserved in the Hans group ( Figure 27 ), suggesting that these interactions may contribute to the dimerization of these lanmodulins.

[0504] Dy III -Hans-LanM structure demonstrates the importance of the second sphere in controlling ligand positioning ( Figure 53 , Figures 28-30 , Table 9-10). Dy III -The overall structure of Hans-LanM is similar to La III -Hans-LanM can overlap a lot, Dy in EF1-3 III The coordination sphere of the ion and La III -Similarity in Hans-LanM ( Figure 3 a inset), but E9 (e.g., Glu91 in EF3) is a notable exception. III The bidentate ion is transformed into a smaller Dy IIIThe monodentate ion results in a nine-coordinate distorted capped square inverse prism geometry; this is consistent with the lower coordination number of the HRE ion in other RE complexes. In EF3, this carboxyl shift shifts the distance between Arg100 and the proximal Oε of Glu91 from (In La III -Hans-LanM) extended to ( Figure 31 ). The rearrangement of this second sphere hydrogen bonding network is K 二聚体 The differences in the RE dependence of the values ​​provide a structural basis.

[0505] The metal binding site of Mex-LanM is fundamentally different from that of Hans-LanM. In Mex-LanM, all four EF hands are composed of nine-coordinated (EF1-3) or ten-coordinated (EF4) Nd III Ion occupancy, each metal binding site includes two solvent ligands, which are absent in Hans-LanM ( Figure 3 b, Figure 32 The observation of two solvent molecules per metal site and a hydrogen bond connecting the D9 residue confirms recent spectroscopic studies. The difference in coordination number between EF1-3 and EF4 is due to the fact that the D3 carboxylates are monodentate in EF1-3 and bidentate in EF4. Although Mex-LanM Nd III Site sharing is in Dy III / La III -Nine / deca-coordinates observed in Hans-LanM, but they are different from the hepta-coordinate Ca II The binding sites are more similar ( Figure 22 The increased coordination number in Mex-LanM relative to calmodulin comes from the bidentate coordination of D5 and the additional solvent ligands. These similarities suggest that LanM has a higher affinity for calmodulin than Calmodulin. II 10 of RE 8 The unique selectivity of the 10-fold is mainly caused by subtle differences in the second coordination sphere and other more distal interactions. Finally, the first coordination sphere in the Hans-LanM protein, especially due to the coordination of E9, produces a more extended hydrogen bonding network ( Figure 24 、 33 ), which likely enhances control over the radius of the binding site. Thus, these structures rationally explain the extraordinary RE / non-RE selectivity of Mex-LanM and Hans-LanM, as well as their differences in LRE / HRE selectivity.

[0506] Single-stage Nd III / Dy IIIThe differences in stability and structure of the LRE and HRE complexes of Hans-LanM suggest that Hans-LanM (wild type and / or R100K) will perform better than Mex-LanM in RE / RE separation. III and Dy III The separation of the RE pairs was the focus of the study. First, the stability of wild-type and R100K-Hans-LanM RE complexes towards citrate, which was previously used as a desorbent for Mex-LanM, was determined. As expected based on the lower affinity, the RE-Hans-LanM complexes were generally less stable towards citrate compared to Mex-LanM ( Figure 1 e), but Nd III -Hans-LanM and Dy III -Hans-LanM complexes (the difference is expressed as the fluorescence of the two Trp residues of Hans-LanM) Figure 34 ) The reported citrate concentration required for 50% desorption of each metal is [citrate] (1 / 2) is also 2 times larger than the Mex-LanM complex ( Figure 4 a, Table 11, Figure 54 ). In addition, the R100K mutation significantly destabilized the La III The stability of the complex to citrate is significantly improved, while that to Nd III The complex has only a slight effect on Dy III The results indicate that dimerization selectively stabilizes the LRE complex of Hans-LanM (especially La III complex), a factor that is abolished by the R100K mutation. Using malonate (a weaker chelator than citrate), Dy III Can be easily desorbed from Hans-LanM and R100K without significant Nd III desorption phenomenon, which indicates that the III / Dy III Separation conditions ( Figure 4 b).

[0507] Although the 2-fold modulation of RE / RE selectivity by dimerization appears small, this difference provides an opportunity to reduce the number of separation stages and improve the efficiency of the separation process. Therefore, as described, Hans-LanM and R100K variants were immobilized on maleimide-functionalized agarose beads via the C-terminal Cys residue and Nd III / Dy IIIUnlike RE in solution and compared to 2 equivalents of Mex-LanM and R100K-Hans-LanM, immobilized Hans-LanM bound about 1 equivalent of RE ( Figure 35 Hans-LanM and R100K showed similar separation capabilities in the La-Gd range (although R100K showed greater Gd-Dy separation capability), as determined by the on-column distribution ratio (D) of the mixed RE solution at equilibrium (Tables 12-14, 19, Figure 4 c). These Nd / Dy separation factors are almost double (Hans-LanM) and triple (R100K-Hans-LanM) those of Mex-LanM (Table 19). Figure 4 As indicated in b, immobilized Hans-LanM was loaded into a model e-waste mixture with 5% dysprosium and 95% neodymium to a break-through energy of 90%, and then eluted with a short, stepwise gradient of malonic acid followed by complete desorption using HCl at pH 1.5. In a single purification stage, the purity of Dy increased from 5% to 83%, and Nd was recovered at a purity of 99.8% (both yields >98%) ( Figure 55 This is significantly better than a comparable process based on Mex-LanM, which only achieved 50% purity in the first separation stage and required a second stage to obtain >98% purity. The immobilized R100K variant performed even better, achieving Dy III and Nd III Baseline separation, purity>98%, yield>99% ( Figure 4 d). The better performance of the R100K variant was unexpected and at this immobilization density, the presence of functional dimers on the column is unlikely (discussed in Figure 55 Thus, although the description of the dimerization mechanism of Hans-LanM leads to substantially improved performance relative to Mex-LanM, full exploitation of the dimerization phenomenon on the column may require tethering both monomers to a single polypeptide chain, a point that is under investigation.

[0508] Conclusions: The 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 recent concepts of dimer-dependent metal recognition emerging from synthetic lanthanide complexes and engineered transition metal-binding proteins and demonstrating that these principles are inherent in nature. This disclosure demonstrates that the strength of dimerization, and thus metal selectivity, can be rationally tuned. Hans-LanM gradually forms LRE-selective dimers at physiological protein concentrations, which are closer to the concentrations used in the biochemical assays disclosed herein (10-20 μM) than to the on-column concentration (approximately 3 mM). Thus, during separations, dimerization can be exploited to its full potential by shifting dimerization sensitivity to higher concentration regimes, such as by fine-tuning hydrophobic interactions at the dimerization interface. Furthermore, these studies establish that lanthanoid proteins with as little as 33% identity exhibit useful differences in metal selectivity. Finally, the exclusive solvent coordination sphere of Hans-LanM should be superior to Mex-LanM in RE / actinide separations, luminescence-based sensing, and stabilization of easily hydrolyzed ions. Further characterization of the coordination and supramolecular principles of biological f-element recognition will inspire the design of ligands with enhanced RE / RE selectivity and their application in novel RE separation processes.

[0509] Bioinformatics methods. a) Protein and genomic sequence data. The sequence of LanM from M. extorquensAM1 was used as the query condition, and PSI-BLAST search was performed against the non-redundant protein sequence (nr) and metagenomic protein (env_nr) databases of the National Center for Biotechnology Information (NCBI) until convergence. Subsequently, the 3047 protein sequences obtained were manually sorted to find those with a length of less than 200 residues, at least one pair of EF hands separated by less than 14 residues, and protein sequences with four EF hands. SignalP (v6.0) was used to predict the signal peptide of the LanM sequence and then removed it before further sequence analysis. b) Construction of sequence similarity network. The similarity between all peptide sequence pairs was calculated using the enzyme function start-enzyme similarity tool, with an E-value threshold of 1×10 -5. Subsequently, a sequence similarity network (SSN) consisting of 696 nodes and 241,853 edges was constructed by Cytoscape (v3.9.1) and explored using an organic layout by Cytoscape (v3.9.1) and subsequently visualized in R (v4.1.0). The edge identity percentage threshold was gradually increased from 40% to 90% to generate distinct clusters. c) Multiple sequence alignment and phylogenetic analysis. LanM sequences were aligned using MUSCLE (v5.1) with default parameters. The model used to build the phylogeny was selected using ModelFinder in IQ-TREE (v2.2.0.3) with --mset set to beast2. A Bayesian phylogeny was generated based on these results using BEAST (v2.6.7). The generated phylogeny was analyzed with 10 7 The first 25% of the initial phase (burn-in) was removed and then visualized using ggtree (v3.2.1).

[0510] Expression and purification of Hans-LanM and its R100K variant. The gene encoding Hans-LanM was obtained from Twist Bioscience, codon-optimized for expression in E. coli and lacking its native 23-residue signal peptide (see Table 15), and inserted into pET-29b(+) using the restriction endonuclease sites NdeI / XhoI. Hans-LanM was overexpressed at a 2 L scale and purified according to the established protocol for Mex-LanM, with one adjustment: after the 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 to remove Ca. II and trace metal impurities. This procedure produced approximately 15 mL of 550 μM protein, which was not further concentrated. A final yield of 45 mg protein per liter of culture medium was obtained. Based on the ExPASy ProtParam tool, 11000 M -1 cm -1 Protein concentration was calculated using the extinction coefficient. R100K-Hans-LanM was purified using the same procedure, yielding 30 mg of protein per liter of culture medium.

[0511] Circular Dichroism (CD) Spectroscopy. Unless otherwise indicated, CD spectra of Hans-LanM were collected at 15 μM (monomer concentration) in Chelex-treated buffer A (20 mM acetic acid, 100 mM KCl, pH 5.0) as described. Buffered metal solutions were prepared as described. Additional details are provided herein.

[0512] Protein samples were prepared for SEC-MALS and SAXS. Samples of wild-type Hans-LanM were prepared by slowly adding 3.0 equivalents of metal (0.5 equivalents at a time, followed by mixing) to 1.0 mL of concentrated Hans-LanM stock solution (550 μM). At these protein concentrations, LRE samples (e.g., La III ) showed slight precipitation, while HRE samples (such as Dy III ) a clear precipitate appeared. The sample was centrifuged at 12,000 × g for 2 minutes to remove the precipitate and then purified using gel filtration chromatography (HiLoad 10 / 300 Superdex 75 pg, 1 mL sample loop, 0.8 mL / min) in buffer B (30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0). The peak containing Hans-LanM was collected (elution volume between 12.0 and 15.0 mL) to avoid the high molecular weight aggregate peak, yielding 2.0 mL of metallated Hans-LanM (1.37-1.53 ​​mg / mL) at concentrations between 114 μM and 128 μM.

[0513] The R100K-Hans-LanM sample did not form high molecular weight species or precipitate upon metal addition. To prepare a sample of this protein, a 500 μM protein solution was diluted to 250 μM (3 mg / mL) in buffer B containing 0.75 mM specific RECl3, yielding a final solution of 3 mg / mL protein at a 1:3 metal ratio for direct analysis by SEC-MALS.

[0514] For calcium conditions, the protein was diluted to 250 μM (3 mg / mL), 5 mM CaCl 2 was added, and the sample was incubated at room temperature for 1 hour. The buffer used for SEC-MALS was the same as above except it also contained 5 mM CaCl 2.

[0515] Online size exclusion chromatography and multi-angle light scattering (SEC-MALS) was performed using an Agilent 1260 Infinity II HPLC system equipped with an autosampler and a fraction collector. The Wyatt SEC hydrophilic column had 5 μm silica beads and a pore size of The dimensions were 7.8×300 mm. A Wyatt Technology DAWNMALS and a Wyatt Optilab refractive index (RI) detector were used to analyze the molar mass of the peaks eluted from the column. The SEC-MALS system was equilibrated with buffer B for 5 hours. Calibration was performed using the same buffer containing bovine serum albumin (BSA, monomer MW: 66 kDa), and the MALS and RI detectors were normalized and aligned. The injection volume for each sample was 15 μL, the flow rate was 0.8 mL / min, and the chromatographic run time was 25 minutes. Data were analyzed using ASTRA software (Wyatt). When SAXS analysis was required, 150 μL of protein (approximately 4 mg / mL) was injected for a second run, and 200 μL fractions of the main peak were collected. BioSAXS data were subsequently collected in triplicate.

[0516] Isothermal Titration Calorimetry. Apo, bound La, and β-lactamase were determined by diluting concentrated protein stocks followed by isothermal titration on a TA Instruments low volume automated affinity ITC. III and combined with Dy III The dissociation constant of the Hans-LanM dimer. The syringe contains 300 μM (apo or 2 equivalents of Dy III Bound) protein or 150 μM or 540 μM (2 equivalents La III The chamber contained 185 μL of matching buffer (30 mM MOPS, 100 mM KCl, pH 7.0) for the bound protein. Titrations were performed at 30°C. Unless otherwise stated, titrations consisted of an initial 0.2 μL injection followed by 17 × 2 μL injections, with a stirring speed of 125 rpm and an equilibrium time of 180 s between injections. Heat was fitted to the "dimer dissociation" model using NanoAnalyze software to produce the dimer dissociation constant (K 二聚体 ), dissociation enthalpy (ΔH) and dissociation entropy (ΔS). All parameters are shown in Table 18.

[0517] K 二聚体 Defined as balance The dissociation constant, K 二聚体 =[M] 2 / [D], where [D] is the concentration of dimers, [M] is the concentration of monomers, and the total protein concentration [P] (measured using the extinction coefficient of the monomer) is given by [P] = [M] + 2[D]. Therefore, K 二聚体 =2[M] 2 / ([P]-[M]) or

[0518] 2[M] 2 +K 二聚体 [M]-K 二聚体[P] = 0 (Formula 1)

[0519] This equation can be used to calculate K from ITC experiments during SEC-MALS experiments. 二聚体 The concentrations of monomers and dimers can be estimated from the [P] values ​​and the SEC-MALS curves. Given the SEC-MALS data, this equation can also be used to estimate the concentrations of La III The maximum possible K for the bound protein 二聚体 .

[0520] BioSAXS. Small angle X-ray scattering (BioSAXS) acquisitions were performed on the RE-complexed Hans-LanM using the equipment and conditions described herein at the protein concentrations listed in Table 5.

[0521] Table 5 lists the forward scattering I(0) and the radius of gyration (R g ) values, which are calculated using the Guinier approximation, which assumes that at very small angles (q<1.3 / R( g )), the intensity is approximately I(q)=I(0)exp[-1 / 3(qR g ) 2 ]. In La III 、Nd (III) and Dy III Under the combined conditions, this agrees with the calculated size of the crystal dimer. The molecular mass was estimated by comparing the SAXS data with the BSA standard. The Guinier R values ​​in the data files were analyzed using ATSAS software. g , maximum particle size (D max ), Guinier fit, Kratky plot, and pairwise distance distribution function. GNOM in ATSAS was used to calculate the pairwise distance distribution function P(r), and from this, R was determined. g and D max The solvent inclusions were calculated using DENSS. The theoretical scattering curves of the constructed model were calculated using CRYSOL and fitted with the experimental scattering data. OLIGOMER was used to estimate the monomer and dimer fractions.

[0522] Prepare protein samples for crystallography. To Hans-LanM (2 mL, 1.16 mM, buffer B), slowly add 3.0 equivalents of LaCl3 or DyCl3, 0.5 equivalents at a time, and mix to reduce precipitation. Remove the precipitate by centrifugation at 12,000 × g for 2 minutes. Remove any soluble aggregates and exchange the protein into a glycerol-deficient buffer (buffer C: 30 mM MOPS, 50 mM KCl, pH 7.0) by gel filtration chromatography (HiLoad 16 / 600 Superdex 75 pg, 1 mL sample loop, 0.75 mL / min). Pool the peaks in the 70-85 mL range and concentrate the fractions to approximately 500 μL for a final concentration of approximately 1.3 mM.

[0523] Mex-LanM was purified as described and exchanged into buffer C before crystallization. The protein was loaded with 3.5 equivalents of Nd III (NdCl3).

[0524] General crystallographic methods. Diffraction datasets were collected at the Life Sciences Collaborative Access Team (LS-CAT) ID-G beamline and processed with the HKL2000 software package. In all structures, phase information was obtained by the single-wavelength anomalous diffraction (SAD) method with phenix.autosol, where lanthanide ions identified with HySS were used as anomalous scatterers. The initial model was generated with phenix.autobuild, followed by multiple rounds of manual modification and refinement in Coot and phenix.refine. In the final stage of model refinement, the anisotropic displacement parameters (ADPs) and occupancies of all lanthanide sites were refined. Model validation was performed with the Molprobity server. Figures were generated using the PyMOL molecular graphics package ( LLC).

[0525] Determination of the structure of Hans-LanM bound to La. Crystals were obtained using the sitting-drop evaporative diffusion method, where 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 plate manufactured by Hampton Research (Cat. No. HR1-002). After three days, thin, flake-like crystals appeared. Crystals suitable for data collection were mounted on rayon loops, briefly immersed in a cryoprotectant solution consisting of well solution supplemented with 10% ethylene glycol, and then snap-frozen in liquid N2.

[0526] Load La IIIThe Hans-LanM was crystallized in the P21 space group (β = 90.024°) with 4 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 in each chain, 12 La III ions (3 per chain in the first, second, and third EF hands), 4 Na I ions (1 per chain in the fourth EF hand), 273 water molecules and 2 citrate molecules. Of the modeled residues, 100% were located in the allowed or preferred regions indicated by Ramachandran statistical analysis.

[0527] Determination of the Hans-LanM structure bound to Dy. Crystals were obtained using the sitting drop vapor diffusion method, where 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) PEG6000 at room temperature in a 24-well plate (Cat. No. HR1-002) manufactured by Hampton Research. Within 1 month, thin flake-like crystals appeared. Crystals suitable for data collection were mounted on rayon loops, briefly immersed in a cryoprotectant solution consisting of a well solution supplemented with perfluoropolyether cryogenic oil (Cat. No. HR2-814) from Hampton Research, and then flash-frozen in liquid N2.

[0528] Load Dy III The Hans-LanM was crystallized in the P21 space group (β = 93.567°) with 4 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 in each chain (except for chain D, where residues 34-38 could not be modeled), 14 Dy III ions (4 in chains A and D, 3 in the second, third, and fourth EF hands of chains B and C), and 656 water molecules. Of the modeled residues, 100% were located in the allowed or preferred regions indicated by Ramachandran statistical analysis. This article describes the collection of the anomalous dataset.

[0529] Determination of the structure of Mex-LanM bound to Nd. Crystals were obtained using the sitting drop evaporation diffusion method, where 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) PEG1500 at room temperature in a 24-well plate manufactured by Hampton Research (Cat. No. HR1-002). Within six months, thin flake-like crystals appeared. Crystals suitable for data collection were mounted on rayon loops, briefly immersed in a cryoprotectant solution consisting of a well solution supplemented with perfluoropolyether cryogenic oil from Hampton Research (Cat. No. HR2-814), and then snap-frozen in liquid N2.

[0530] Load Nd III Mex-LanM was crystallized in the P212121 space group with one monomer in the ASU. The initial FOM and Bayesian CC were 0.799 and 0.56, respectively. The final model consisted of residues 29-133, 4 Nd III ions and 171 water molecules. Of the modeled residues, 100% were located in the allowed or preferred regions indicated by the Ramachandran statistical analysis.

[0531] Fluorescence Spectroscopy. All fluorescence data were collected using a Fluorolog-QM fluorimeter (Horiba Scientific) configured as 75-21-C, equipped with a dual monochromator on the excitation arm and a single monochromator on 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 10-mm quartz spectrofluorimetry cuvette (Starna Cells, 18F-Q-10-GL14-S) was used to collect data at 90° relative to the excitation path.

[0532] Fluorescence lifetime measurements were performed using established methods. Briefly, a 100% H2O matrix (buffer: 25 mM HEPES, 75 mM KCl, pH 7.0) containing Hans-LanM and 2 equivalents of Eu III The protein solution was prepared by lyophilizing the protein solution of 100% H2O and about 99% D2O to obtain a total volume of 4.5 mL. Half of the initial protein mixture (2.25 mL) was retained for future use, while the remainder was lyophilized to remove H2O and resuspended twice with 99.9% D2O to exchange into D2O. The resulting protein solution (100% H2O and about 99% D2O) was mixed in various proportions to produce solutions with a D2O content of 0%, 25%, 50% and 75%. The protein concentration was 20 μM. For each sample, the luminescence decay time constant (τ), λ, was measured with 5000 excitations over a time span of 2500 μs. ex =394nm,λem =615 nm. τ was determined by single exponential fitting using Felix FL Powerfit-10 software (Horiba Scientific). 1 / τ was plotted against the percentage composition of D2O, and the slope (m) of the resulting line was determined. The q value was determined using the following equation (Equation 2):

[0533] q= 1.11[τ -1 H2O - τ -1 D2O - 0.31+ 0.45n OH + 0.99n NH + 0.075n O-CNH ]. (Formula 2)

[0534] Among them, τ -1 H2O and τ -1 D2O The reciprocal of the time constant in 100% H2O and D2O, respectively (the latter is extrapolated using the fitted line equation), in ms -1 ; According to the Hans-LanM crystal structure, n OH =0,n NH =0,n O-CNH =1 (resulting from the metal-coordinated Asn residue). This equation simplifies to formula 3:

[0535] q = 1.11[-m - 0.31 + 0.075] (Equation 3)

[0536] For the fluorescence competition experiments, 20 μM solutions of Hans-LanM or R100K variants were prepared in buffer A (pH 5.0) containing 2 equivalents of metal (40 μM). Fluorescence emission spectra were collected using the following settings: ex =278nm,λ em =300-420 nm, integration time = 0.5 s, step size = 1 nm. Titration was performed by adding at least 0.6 μL of titrant (from concentrated stock solutions of 10 mM-1 M citrate or malonate, pH 5.0). Spectra were corrected for dilution. Each experiment was performed in triplicate.

[0537] Purification of Cysteine-Containing Variants. R100K-Hans-LanM-Cys was expressed and purified as described for Mex-LanM-Cys, with a final yield of 50 mg protein per liter of culture medium. For Hans-LanM-Cys, the purification procedure followed the same modified protocol as described for Mex-LanM-Cys (minus the dialysis step), except that the SEC step was run using a reducing buffer (30 mM MOPS, 100 mM KCl, 5 mM TCEP, pH 7.0) containing 5 mM EDTA and was frozen under liquid N2 prior to immobilization.

[0538] Maleimide functionalization of agarose beads Amine-functionalized agarose beads were maleimide functionalized as described previously.

[0539] Immobilization of Hans-LanM and R100K variants. Immobilization of R100K-Hans-LanM was performed by thiol-maleimide conjugation reaction as previously described. For Hans-LanM, a final protein concentration of approximately 0.4 mM (8 mL) was mixed with 1 mL of maleimide microbeads and the conjugation reaction was carried out at room temperature for 16 hours. 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, a Pierce TM BCA protein assay (Thermo Fisher Scientific) was used to determine the LanM concentration in the reaction solution before and after the conjugation reaction.

[0540] Batch experiments were performed to determine the separation factor. The LanM-immobilized beads were washed with deionized water. Feed solution (5 mL, equimolar RE La-Dy, total 3 mM, pH 5.0) was added to 1 mL of beads and incubated for 2 hours. The equilibrium solution was collected and the concentration of RE was determined by ICP-MS as [M]. ad . RE was then desorbed from the microbeads using 4 mL of 0.1 M HCl, and its concentration was determined by ICP-MS, i.e. [M] de .

[0541] The RE distribution coefficient (D) between the LanM phase and the solution phase was calculated as:

[0542] D=[M] LanM / [M] 液体 (Formula 4)

[0543] Among them, [M] LanM and [M] 液体are the molar concentrations of the metal ions in the LanM phase and the solution phase at equilibrium, respectively. To account for the free liquid absorbed by the agarose beads, the following correction was used: [M] 液体 =[M] ad ;

[0544] [M] LanM =(4×[M] de -[M] ad ) / 4.

[0545] The separation factor (SF) is defined as follows:

[0546] SF = D RE1 / D RE2 (Formula 5)

[0547] Among them, D RE1 and D RE2 are the distribution coefficients of RE1 and RE2 respectively.

[0548] Leak-through column experiments. The columns were packed, run, and analyzed for metal concentrations as described in our previous work; details are included in this article.

[0549] In RE paired separation experiments, the purity and yield of metal ions are defined as follows:

[0550] Purity (RE1) = C RE1 / (C RE1 +C RE2 ) (Formula 6)

[0551] Yield (RE1) = (RE1 recovered) / (total amount of RE1 loaded) (Equation 7)

[0552] Among them C RE1 and C RE2 are the molar concentrations of RE1 and RE2, respectively.

[0553] Table 17. Characterization of Hans- and Mex-LanM metal complexes using SEC-MALS. The concentrations of protein samples loaded onto the column were: 1.2-1.5 mg / mL for apo and RE-bound Hans-LanM, II The bound amount for Hans-LanM was 3 mg / mL and for Mex-LanM was 3 mg / mL. For samples containing RE, the protein was mixed with 3 equivalents of the corresponding RE. III Ion preincubation. For Ca II5 mM CaCl2 was added to the running buffer. The apoprotein eluted as two peaks, the first being a minor contribution peak (10% of the protein, 56.5 kDa) and the second being a major peak (90% of the protein, 12.9 kDa). For full details of sample preparation, see "Materials and Methods." The Hans-LanM values ​​were plotted on Figure 2 a; Figure 9 The raw data of La, Nd and Dy are shown in .

[0554]

[0555]

[0556] Table 18. ITC thermograms obtained by fitting the dimer dissociation model for apo and Dy III Thermodynamic parameters of the bound Hans-LanM. III For proteins, these values ​​could not be determined because no changes in the measured heat were observed during the titration experiments ( Figure 12 ). The reported values ​​are the means of three independent titrations with standard deviations.

[0557]

[0558] Table 19. Distribution factors (D) and separation factors (SF) for binary Nd / Dy solutions equilibrated using a Hans-LanM column or a R100K-Hans-LanM column. 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 its composition was determined by ICP-MS analysis to be 1.42 (4) mM Nd and 1.62 (32) mM Dy. The pH was 5.0. This experiment demonstrates that the immobilized R100K variant exhibits improved on-column separation characteristics compared to the wild-type Hans-LanM. Details of the uncertainty values ​​in parentheses are provided in the legend to Table S12.

[0559]

[0560] General Considerations. Unless otherwise stated, chemical reagents were obtained from Millipore Sigma at the highest purity. Chemical Compositions E. coli BL21 (DE3) cells were from New England Biolabs. Biochemical and column-based experiments were performed using RE chloride salts and buffers obtained from MilliporeSigma with a purity of at least 99.9%. Anion exchange chromatography was performed using Q Sepharose Fast Flow resin from MilliporeSigma. Automated protein chromatography was performed on a GE Healthcare Biosciences Akta Pure fast protein liquid chromatography (FPLC) system using a HiLoad Superdex 75 pg 16 / 600 column at preparative scale and a Superdex 75 pg Increase 10 / 300 GL column at analytical scale. Quantification of RE was performed using an inductively coupled plasma mass spectrometer (ICP-MS; Thermo Scientific iCAP RQ) using helium in KED mode. The ICP-MS was located at the Laboratory for Isotopes and Metals in the Environment (LIME) at the Institute for Earth and Environmental Systems Research, Pennsylvania State University. For protein immobilization, amine-functionalized agarose beads were purchased from Nanocs Inc. N-Succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was purchased from Chem-Impex International Ltd. and used without further purification.

[0561] Circular dichroism (CD) spectroscopy. As previously described, collect the CD spectrum of Hans-LanM. In brief, use JascoJ-1500CD spectrometer, scan sample in the range of 195 to 255nm, be set as follows: 1nm bandwidth, 0.5nm data spacing, 50nm / min scan rate, 4s averaging time. For all buffered metal titrations and stoichiometric titrations, 15 μM protein (monomer concentration) is contained in the cuvette. When carrying out stoichiometric titration, protein is diluted into buffer A (20mM acetic acid, 100mM KCl, pH 5.0) processed through Chelex, and titrated with 1.5mM metal ion solution (each metal ion 0.5-4.0 equivalent) in the same buffer.

[0562] The buffered metal solutions were prepared as described previously. II 、La III and Nd III When titrating Dy IIIEGTA was used as a chelating agent. For each metal ion, 15 μM of protein was added to the respective "high" and "low" metal solutions. These solutions were then combined in varying ratios to a final volume of 200 μL to generate 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 to generate binding curves fitted using the Hill equation.

[0563] BioSAXS data collection. Data were acquired at the Penn State X-ray Crystallography Facility using the primary source. The X-rays were collected at 100 nm wavelength using a Rigaku MM007 rotating anode equipped with a BioSAXS2000 nano-Kratky camera system. The system includes the OptiSAXS confocal maximum flux optical system 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 GemDugout, State College, PA). The useful q-space range (4πsinθ / λ, where 2θ is the scattering angle) is typically from arrive The energy of the X-ray beam was 1.2 keV, the Kratky block attenuation was 22%, and the beam diameter was approximately 100 μm.

[0564] The protein sample was loaded into a quartz capillary flow cell using an autosampler, which was mounted on a platform maintained at 22°C and aligned with the X-ray beam. The sample chamber and the entire X-ray flight path (including the beam stop) were kept under vacuum (<1×10 -3 The data of each protein sample and matching buffer solution were collected in a 1M NaOH container (500 μm) to eliminate air scattering. Rigaku SAXSL AB software was programmed to automatically collect the data of each protein sample and matching buffer solution, and was rigorously cleaned with 1M NaOH before the start of the operation, and was cleaned with water and ethanol between multiple sample runs. Data reduction (including image integration and normalization) and background buffer solution data subtraction were also performed using SAXSLAB software. After ensuring that no X-ray radiation damage occurred, six 10-minute images were collected from the protein and buffer solution samples and averaged. SAXS data superposition shows that no radiation decay occurred during the 60-minute data acquisition process. Subtract reference buffer solution subsequently to obtain the original SAXS scattering curve only from protein.

[0565] The anomalous scattering data set was collected at beamline ID-D at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL) at the National Cancer Institute Structural Biology Facility (GM / CA). The X-ray absorption spectrum ( Figure 29 ), and accordingly selected 7793.5eV(L III Scattering data at these two energies were collected alternately using a reverse beam geometry with a 30-degree wedge to minimize radiation damage and subsequently processed using HKL2000.

[0566] The coordinates determined from the high-resolution data were further refined with a new data set 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, while the remaining 13 dysprosium ions were retained. No other significant structural changes were observed. The anomaly difference map was drawn using phenix.maps and the corresponding peak intensities were checked by Coot (Table 10). The large difference in the intensities of the anomaly peaks determined at the energies of the front and edge corresponding to Dy strongly suggests that the metal bound to the EF hand is indeed Dy.

[0567] Maleimide functionalization of agarose beads. Maleimide functionalization of amine-functionalized agarose beads was performed as previously described. In short, agarose microbeads (1.2 mL) were aliquoted into 5 mL Eppendorf tubes and pre-conditioned with phosphate buffered saline (PBS) at pH 7.4. The final volume after resuspension was approximately 1.7 mL (1.2 mL microbeads and 0.5 mL PBS supernatant). SMCC (0.15 gram) powder was dissolved in 3.4 mL DMSO and mixed with microbeads. After incubation at room temperature for 2.5 hours on a shaking mixer, the modified agarose microbeads were washed three times with DMSO to remove unreacted SMCC and subsequently washed three times with coupling buffer (50 mM HEPES, 50 mM KCl, pH 7.0) to remove DMSO. Maleimide microbeads were subsequently used for LanM immobilization within 2 hours.

[0568] Leakage 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 added gravimetrically. Prior to leakage experiments, the column was washed with 25 mM HCl and MilliQ water, and conditioned with buffer D (10 mM isopiperazine-1,4-bis(2-ethanesulfonic acid), pH 5.0). RE stock solutions were prepared by dissolving individual RE chloride salts in 1 mM HCl. The stock solutions were diluted in buffer D. Unless otherwise stated, RE solutions were pumped at a rate of 0.5 mL / min, and column effluent was collected in 1.0 mL aliquots. Prior to desorption experiments with the indicated concentrations of chelating agent or hydrochloric acid, the column was washed with 5 bed volumes of MilliQ water. For single RE ion solutions, the arsenazo III assay was used to quantify the RE ion concentration. Specifically, 40 μL of sample was mixed with 40 μL of 12.5 wt.% trichloroacetic acid (TCA) and then added to 120 μL of a 0.1 wt.% filtered arsenazo solution in 6.25 wt.% TCA. The absorbance at 652 nm was measured and compared with a standard to determine the RE metal ion concentration. ICP-MS also demonstrated the accuracy of the colorimetric method. For experiments with RE mixtures, the metal ion concentration was determined by ICP-MS (Table 19, Table 16).

[0569] Table 1. ICP-MS analysis of metallated Hans-LanM samples used for SAXS or crystallographic analysis. Samples for SAXS analysis were incubated with 3 equivalents of metal ion, and the soluble protein was run on an S75 SEC column to remove aggregates. Sample preparation for crystallographic analysis was described herein. The lower metal loading in samples used for crystallographic studies may be related to the additional spin concentration step and the presence of a third equivalent binding to a relatively weak site. Data are presented as the mean of three technical replicates with s.d.

[0570]

[0571] Table 2. Hans-LanM and La III 、Nd III 、Dy III and Ca II Summary of fitting parameters for CD titration. Apparent K d (K d,app ) values, Hill coefficient (n), and change in molar ellipticity at 222 nm (Δ[Θ]) are reported as the mean ± SEM of the fitted values ​​from three independent titrations. Conditions: 15 μM monomer concentration, 25°C, 20 mM acetic acid, 100 mM KCl, pH 5.0.

[0572]

[0573] Table 3. RE from analytical SEC (Superdex S75) III -The apparent molecular weight of the Hans-LanM complex. The apparent molecular weight value is derived from Figure 8 , also drawn in Figure 2 a. Compare with the SEC-MALS data in Table 17. The ionic radii of the previously described decacoordinate (La) and nine-coordinate (Nd-Ho) complexes are given. For experimental details, see Figure 8 legend.

[0574]

[0575] aCN=10. bCN=9.

[0576] Table 4. Data collection and refinement statistics for the X-ray structures of La-Hans-LanM, Dy-Hans-LanM, and Nd-Mex-LanM. The statistics for the highest resolution shell are shown in brackets.

[0577]

[0578]

[0579] Table 5. SAXS structural parameters of Hans-LanM in the presence of different metal ions. Data were acquired by in-house RigakuBioSAXS2000. nano Collect. Buffer conditions: 30 mM MOPS, 100 mM KCl, 5% glycerol, pH 7.0. Porod volume analysis supports the conclusion that the La and Nd complexes of Hans-LanM are mainly dimeric, while the volume of the Dy complex is only about 2 / 3 of that of the La and Nd complex, which is an equilibrium mixture of monomers and dimers. Note that the R g The differences between the values ​​are smaller than might be expected based on SEC-MALS (Table 17 shows that the hydrodynamic radii of La / Nd and Dy differ by approximately ), this is because the protein concentration used in SAXS is 5-fold higher than that used in SEC-MALS, so the population of Dy-bound dimers is substantially larger in the SAXS experiment. Nevertheless, these RE-dependent differences are still observed in the SAXS R g The uncertainty of the value (see Figure 15 ).

[0580]

[0581] Table 6. Analyses using OLIGOMER and CRYSOL software indicate that the La and Nd complexes of Hans-LanM are almost entirely dimerized, but the Dy complex is a mixture of monomers and dimers. The OLIGOMER program fits the experimental SAXS scattering curves of multicomponent protein mixtures to determine the volume fractions of each component in the mixture. CRYSOL evaluates the solution scattering of macromolecules with known atomic structures and fits them to the experimental scattering curves. Both analyses were performed in two possible states: La-Hans-LanM crystal dimers and crystal monomers. This analysis indicates that the volume fractions of the La-Hans-LanM and Nd-Hans-LanM complexes are preferentially dimers, which is very likely close to the true distribution due to the availability of crystallographic dimer models. OLIGOMER and CRYSOL analyses of the Dy case indicate an equilibrium between dimers and monomers.

[0582]

[0583] Table 7. R100K-Hans-LanM and La III 、Nd III and Dy III Summary of CD titration fitting parameters. Apparent K d (K d,app ) values, Hill coefficient (n), and change in 222 nm molar ellipticity (Δ[Θ]) are reported as the mean ± sem of the fits from two (Nd, Dy) or three (La) independent titrations. Conditions: 15 μM monomer concentration, 25°C, 20 mM acetic acid, 100 mM KCl, pH 5.0.

[0584]

[0585] Table 8. SEC-MALS analysis of R100K-Hans-LanM. The protein sample concentration loaded onto the column was 3 mg / mL (3.5-fold higher than the wild-type Hans-LanM sample concentration).

[0586]

[0587] aND, not determined

[0588] Table 9. L at 7760.0 (front edge) and 7793.5 eV (Dy III Data collection statistics for the Dy-Hans-LanM anomalous diffraction data set collected at the edge of the image. Statistics for the highest resolution shell are shown in parentheses.

[0589]

[0590]

[0591] Table 10. Dy-Hans-LanM at 7760.0 (front edge) and 7793.5 eV (Dy L III The abnormal peak height of the edge) is in Interestingly, EF2 and EF3 exhibited the largest aberrant difference plot peaks, perhaps reflecting the biochemical observation that there are only two high-affinity sites in this complex ( Figure 1 d, Figure 6 N / A: Not applicable. *No heavy elements were found in EF1 of the D chain in this crystal.

[0592]

[0593] Table 11. Desorption of 50% La from Hans-LanM and R100K-Hans LanM III 、Nd III 、Dy III Required citrate and malonate concentrations. See Figure 4 a and Figure 54 The Trp emission intensity of Hans-LanM was monitored at 333 nm. Initial conditions: 20 μM protein, 40 μM RE, 20 mM acetic acid, 100 mM KCl, pH 5.0, to which increasing concentrations of citrate or malonate were added dropwise. Data represent the mean ± sem (in parentheses) of three independent titrations.

[0594]

[0595] a 350 mM is the highest concentration tested. Figure 4 b.

[0596] Table 12. Distribution factors (D, bold) and separation factors (SF) of selected REs immobilized on Mex-LanM. The D values ​​represent the distribution of specific metal ions between LanM and solution in multielement equilibrium experiments described in the main text and in Materials and Methods ("Batch experiments to determine separation factors"), where 5 mL of feed solution of equimolar REs from La to Dy (total RE of 3 mM, total volume of 15 μmol, pH 5.0) was equilibrated with 1 mL of immobilized LanM microbeads (capacity: 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 of LanM. The separation factor as a function of the identity of the RE was calculated as D metal(top) / D metal(left)An SF value of 1.0 indicates no selectivity within the RE, while an SF value much greater than (or less than) 1 indicates a preference for a particular ion over other ions. A common logarithmic plot of the D value for each metal ion is shown in Figure 4 c. Uncertainties are shown in brackets, where the number in brackets is the uncertainty of the immediately preceding 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 those bound to Hans-LanM and R100K-Hans-LanM, the uncertainties are larger because the weaker binding to LanM compared to LREs means that the amount of RE adsorbed on the column is very small. The uncertainty in the D value is the standard deviation of three independent column runs. The uncertainty in the SF value is calculated by propagating the error of the corresponding D value.

[0597]

[0598] Table 13. Distribution factors (D) and separation factors (SF) of selected REs immobilized with Hans-LanM. See legend to Table 12 for details.

[0599]

[0600] Table 14. Distribution factors (D) and separation factors (SF) of selected REs immobilized with R100K-Hans-LanM. See the legend to Table 12 for details.

[0601]

[0602]

[0603] Table 15. Amino acid and DNA sequences of constructs used in this study. The first residue of the cytosolically expressed Hans-LanM protein (after N-terminal Met cleavage) is A24, the signal peptide cleavage site predicted by SignalP 6.0; all residues are numbered according to the full-length sequence.

[0604]

[0605]

[0606] Table 16. Figure 4 d and Figure 55 Metal ion concentrations in the synthetic feed solution for on-column Nd / Dy separation. Concentrations were determined by ICP-MS analysis.

[0607]

[0608] Example 2

[0609] This example describes the use of the peptides / proteins of the present disclosure.

[0610] The structures of Hans-LanM and Mex-LanM and the understanding of the dimerization mechanism of Hans-LanM suggest several avenues for improving metal binding selectivity, stoichiometry, and cooperativity, as well as for modulating K 二聚体 Overall, this places the dimerization equilibrium in a pathway that is more suitable for industrial processes rather than high-concentration systems in cells.

[0611] By identifying points in the lanthanide series where the dimerization propensity changes significantly (at these points, the complex with one lanthanide(III) ion (e.g., Tb) is substantially more stable than the next lanthanide ion (e.g., Dy), the additional stabilization of the metal-protein complex by dimerization can be exploited to improve selectivity. Since the position of the dimerization equilibrium will depend on the monomer concentration, the K can be tuned. 二聚体 It is important to reflect the protein concentration under the conditions of intended use (in most cases, this will weaken the dimerization affinity).

[0612] Increasing the stoichiometry will allow for more economical REE incorporation (better atom economy), while improving the cooperativity will result in sharper desorption profiles, potentially leading to better separations.

[0613] Improved potential separation using Hans-LanM. The response of Hans-LanM to Sm(III), Gd(III), Tb(III), and Ho(III) was determined using CD spectroscopy to fill Figure 1 e and selectivity plots in Table 2 ( Figure 37 , Table 20). The experimental conditions were the same as above (15 μM Hans-LanM in 30 mM acetic acid, 100 mM KCl, 10 mM EGTA, 0-10 mM Ln(III), pH 5.0).

[0614] Table 20. Extension of Table 2 to include Sm, Gd, Tb, and Ho. Reported values ​​and uncertainties are at least twice.

[0615] Metal ions <![CDATA[K d,app ]]> n <]]> <![CDATA[La III ]]> 68(7)pM 1.8(6) -728(57) <![CDATA[Nd III ]]> 91(6)pM 2.0(2) -722(20) <![CDATA[Sm III ]]> 240(40)pM 1.3(2) -575(89) <![CDATA[Gd III ]]> 397pM 1.97 -512 <![CDATA[Tb III ]]> 583pM 1.39 -476 <![CDATA[Dy III ]]> 2600(700)pM 1.3(4) -449(56) <![CDATA[Ho III ]]> 3500(210)pM 1.2(1) -481(76) <![CDATA[Ca II ]]> 60(10)μM 1.0(2) -545(41)

[0616] These data are consistent with the general trend noted above, namely that the magnitude of the protein response to Dy(III) (Δ[Θ]) is smaller than that to La(III) and Nd(III); this reduction in magnitude occurs roughly with Sm(III). This is also consistent with the apparent K d The sudden drop (2.5-3 times) between Nd(III) and Sm(III) is related to the apparent K dThe second sharp drop (5-fold) occurs between Tb(III) and Dy(III), which would represent an industrially important separation. It is unclear whether the approximately 2 Hill coefficients for La(III)- and Nd(III)-bound Hans-LanM (Table 2) reflect metal binding to EF3 and dimerization of two monomers, or a single monomeric EF2 / 3. One explanation for the lower cooperativity of Dy(III) (and other MREE-HREE) binding is that it is indeed metal-dependent dimerization that drives the cooperativity. These results motivate the exploration of exploiting the dimerization equilibrium of this protein variant for such separations.

[0617] For this reason, it is very important to understand the interactions that contribute to dimerization stability. The crystal structure of Hans-LanM shows that the hydrogen bonding network centered on R100 is a key contributor, but other interactions on the interface can also play a role (such as M92, I43, I47, L96). The ITC analysis (Table 21) of the R100K variant supports this viewpoint. Although the presence of glycerol in these experiments can affect the quantified thermodynamic values ​​to a certain extent, the data show that in R100K, the K of the La (III) complex is 二聚体 The values ​​increased significantly from <0.4 μM to 14 μM, while the K values ​​of R100K bound to apo and Dy(III) were 二聚体 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.

[0618] Table 21. Thermodynamic parameters of WT and R100K Hans-LanM obtained by fitting the ITC thermograms to a dimer dissociation model. These experiments were similar to those shown in Table 18, except that the buffer contained 5% glycerol.

[0619]

[0620] Therefore, to make the dimerization equilibrium work at millimolar protein concentrations (required for separation), some of the other interactions that lead to dimerization should be removed. To achieve this, some of the following variants will be used (alone and possibly in combination):

[0621] M92L, M92A, or M92D: LanM_012 contains a leucine at this position, and in the absence of REEs, it does not show significant dimerization (see below). These mutations may reduce the hydrophobicity of the central portion of the dimer interface, thereby reducing the fraction of residues that are clearly involved in Hans-LanM dimerization as observed in ITC and other studies.

[0622] A44N, A44S, or A44T: These mutations are hypothesized to add a hydrogen bond between this residue in one monomer and the T7 residue in EF2 or EF3 of the adjacent monomer. If this interaction is with EF2, this would allow specific interactions with EF2 and EF3 in the dimer, thereby linking dimerization to the occupancy of EF2 and EF3 and potentially enhancing cooperativity and decreased affinity (e.g., Figure 37 observed in ).

[0623] D93N, D93A, or D93E: The D93N or D93A mutations are likely to weaken the interaction of R100 with neighboring monomers, such that only / primarily hydrogen bonding with metal ligands contributes to dimerization in this region of the protein. D93E could alter the selectivity of REE-induced dimerization by altering the hydrogen bonding network, thereby shifting the REE that dimerizes most strongly.

[0624] To immobilize a version of Hans-LanM capable of dimerization on the column, a linker (e.g., a peptide sequence) of appropriate length and flexibility can be used to connect the two monomers. The following are examples of potential constructs (the first one is likely too short to support intramolecular dimerization; the linker sequence is underlined, and the remaining sequence is the Hans-LanM sequence):

[0625] >HansR100K-L1

[0626]

[0627] >HansR100K-L2

[0628]

[0629]

[0630] >HansR100K-L3

[0631]

[0632] >HansR100K-L4

[0633]

[0634] >HansR100K-L5

[0635]

[0636] Dimerization variants and linker variants can be combined to perform separation experiments at higher protein concentrations, either on a column or in solution.

[0637] 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., ACSCent. Sci. 2021) showed that the third binding equivalent assigned to EF1 is kinetically more unstable than the first two, and exhibits a binding stoichiometry of 2 under low pH and column conditions. For example, at pH 5, EF hands 2 and 3 respond to Nd 3+ The apparent K of the conformational change d was 21 pM, while the apparent K d The affinity of EF1 is 4.1 nM (Mattocks et al., Chem. Sci. 2022). Therefore, we sought a way to align the affinity of EF1 with that of EF2 / 3, thereby increasing the stoichiometry to (at least) 3. A general approach is to insert unique hydrogen bonds between the EF hands of the folded protein, which can enhance affinity.

[0638] The crystal structure of Nd-Mex-LanM shows a hydrogen bond between D56 (the residue at the "-3" position of EF2, with the minus sign indicating that it is located before the loop) and K93 (the 10th residue of EF3). At the same time, there is no equivalent interaction between the EF1 / 4 pair (A32 at the -3 position of EF1 and A117, the 10th residue of EF4). We speculate that the placement of an equivalent hydrogen bond in this pair may help stabilize EF1. The substitutions A117K and A117R were made in the context of A32D, and these two proteins (A32D / A117K and A32D / A177R) were expressed and purified from E. coli like wild-type Mex-LanM. The interactions of these proteins with Nd were characterized by CD at pH 5.0. 3+ response to allow direct comparison with wild-type data (Mattocks et al., Chem. Sci. 2022) ( Figure 39 and 40 ).

[0639] Although both sets of substitutions reduced the apparent K d , but the A32D / A117K variants seem to perform better. In the A32D / A117R, the overall response ( Figure 40 , right) is slightly asymmetric, suggesting that EF1 exhibits an enhanced response compared to wild type (see Figure 2), but not similarly to EF2 / 3 in A32D / A117K. In A32D / A117K, based on the value of the Hill coefficient, EF1 likely does not respond cooperatively with EF2 / 3; it is unclear whether the enhanced response is sufficient to increase the stoichiometry of binding or whether this site is still kinetically less stable than EF2 / 3. Although initial xylenol orange competition experiments indicated that A32D / A117R destabilized the overall equivalents of metal binding to some extent, A32D / A117K was similar to WT, and I42L / N108D / I115L was slightly more stable relative to WT ( Figure 38 , bottom), but replication of these results showed that A32D / A117R and A32D / A117K performed similarly and to some extent better than WT (see below and Figure 68 The stoichiometry of binding will be tested using spin columns to separate bound and unbound metal ions at different pH values ​​and using Cys-modified versions immobilized on agarose beads (as previously described). Combinations of the best variants (e.g., A32D / A117K and I42L / N108D / I115L) will also be tested similarly.

[0640] In the crystal structure with almost the same EF ring structure, EF4 was observed to be Nd 3+ The structure reveals a slight unwinding of the helix between EF3 and EF4 starting at A98, likely due to EF4's occupancy. Adding a helical break in this region (i.e., substituting A98, A99, V100, or possibly A102 with Gly) both optimizes the structure of EF4 and decouples it from EF3, potentially providing sufficient conformational change to increase affinity. Similarly, a small break in the helix between EF1 and EF2 at G51 could explain why EF1 behaves largely independently of EF2. The G51A substitution, which links EP1 to EF2, could increase the Hill coefficient to 3. The effects of these mutations were measured by the fluorescence of Y96 at approximately pH 5, which decreases after the addition of the first two binding equivalents but increases after the third equivalent, as if the third equivalent slightly destabilizes the protein fold. Mutations that maintain lower Tyr fluorescence emission upon addition of a third equivalent will be explored.

[0641] Combining these mutations in both helices with the A32D / A117K and / or I42L / N108D / I115L variants described above could bring the tight binding stoichiometry to 4 and link all four EF hands into a single cooperative unit (n up to 4), which could significantly improve the separation of adjacent REs. If this is not possible, activation of EF4 and cooperative binding with EF1 could produce two half-proteins, each with n = 2, which would still significantly improve the protein's performance.

[0642] The concept of hydrogen bonding EF1 and EF4 is to use disulfide. Figure 27 As described in , natural LanM has this property and the metal binding properties of these proteins will be investigated. An example of such a protein has the following sequence (after cleavage of the putative signal sequence; EF hands are underlined):

[0643] >LanM_011

[0644]

[0645] Compared with LanM_001, LanM_002 has a III 、Eu III 、Tb III and Dy III The luminescence of Eu III The luminescence lifetime experiment observed that Hans-LanM (LanM_002) has a zero-coordinated water molecule, and combined with the presence of two Trp residues near EF2 / 3 confirmed by X-ray structure, it suggests that it may not only sensitize Eu III It can also sensitize ions with shorter luminescence lifetimes, such as Sm III and Dy III ( Figures 41-43 ).like Figure 41 and Figure 42 As shown, Hans-LanM can sensitize Eu(III) and Tb(III) under time-resolved and steady-state conditions, while Mex-LanM performs worse. Figure 43 The results show that Sm(III) and Dy(III) luminescence is very poor when bound to Mex-LanM, while a signal can be detected by Hans-LanM. This enables sensitive detection of these metals in a complex environment (similar to the detection of Tb using Trp-LanM) and even within cells.

[0646] Characterization of Flavobacterium LanM (LanM_012). Sequence alignment and structure of LanM in combination prompted us to search for LanM proteins that might exhibit additional hydrogen bonding interactions between other EF hands, which could increase the sensitivity of the protein to lanthanides during dimerization, alter the strength of dimerization, and / or increase cooperativity due to more interconnected EF hands. In the crystal structure, EF3 in one monomer appears to be primarily associated with the occupancy of EF3 in the other monomer (EF4 is also present, but it is blocked by Na + Position occupied). H25 (the third residue after EF1) and T40 (the sixth residue of EF2) interact through the hydrophobic interaction between the imidazole ring of His and the methyl group of Thr. Converting this interaction into hydrogen bonding can provide greater stability. In the X. flavus sequence, H25 is Asp and T40 is Lys, indicating that they can undergo hydrogen bonding. The equivalent T29 is Lys, which can also participate in hydrogen bonding. The peptide sequence of LanM_012 for cytoplasmic expression (signal peptide removed, residues equivalent to H25 and T40 in Hans-LanM are underlined) is:

[0647] >LanM_012

[0648]

[0649] The gene sequence of LanM_012 was inserted into pET29a, like Hans-LanM.

[0650]

[0651]

[0652] The expression and purification of LanM_012 were similar to those of Mex-LanM, yielding approximately 10 mg / L. Due to the N-terminal rule, the addition of Ala as the first residue after the N-terminal Met can increase expression. Figure 44 The results show that while the apoprotein eluted from the SEC column with a retention volume consistent with a monomer, the La and Dy complex eluted as a distinct dimer. This protein's dimerization propensity suggests that the presence of R100 equivalent residues is highly likely to predict the dimerization ability of LanM, although other, currently unknown interactions may contribute to the overall dimerization strength and differences in dimerization propensity between apo- and REE-binding proteins.

[0653] Like Hans-LanM, the third binding equivalent in LanM_012 is relatively weak and cannot surpass xylenol orange ( Figure 45)。However, we found that at room temperature, apo-LanM_012 has helical properties comparable to the LRE-bound states of Hans-LanM and Mex-LanM( Figure 46 ).

[0654] Although the apoprotein has a distinct secondary structure at room temperature, temperature-dependent CD experiments showed that the stability of the helix is closely related to temperature and the presence of metal, and the denaturation temperature increases in the order apo<Dy<La, indicating an increase in the stability of the LREE complex( Figure 47 ). This suggests that it may be possible to selectively desorb HREE from the protein using high temperature.

[0655] Titration of the protein with buffered metal solutions and competitive titration with citrate( Figure 48 , left and right) showed that LanM_012 binds REE more tightly than Hans-LanM. Interestingly, as shown by the fitting parameters related to these data (Tables 22 and 23), the binding of both La(III) and Dy(III) is cooperative (n~2), which may be due to complete dimerization in the presence of both ions( Figure 44 ; compared to Hans-LanM, which is mainly monomeric under these experimental conditions). However, it is noteworthy that the ΔF values of Nd and Dy are the same, while different from La, indicating a conformational difference between the La-binding protein and the Nd-binding protein, and this difference may be exploited to separate LREE (Table 23).

[0656] Table 22. Figure 48 (Left) fitting parameters

[0657]

[0658] Table 23. Figure 48 (Right) fitting parameters

[0659]

[0660] Finally, the similarity between the EF-hand sequences of LanM_012 and Hans-LanM suggests that LanM_012 may also not have a coordinating solvent, which would result in stronger luminescence and higher affinity for the REE complex, which is desirable for sensing applications. Figure 49 The results shown in verify this expectation. In turn, these data also predict that Glu present at position 9 of the EF-hand (perhaps in combination with Asn at position 1) can produce a REE compound without a coordinating solvent.

[0661] Sequence. (Other sequences are shown in Table 15).

[0662] >Hans-LanM (full protein sequence, signal peptide is underlined)

[0663]

[0664] >Hans-LanM (signal peptide removed, as expressed in this study)

[0665]

[0666] >Hans-LanM(R100K) (signal peptide removed, as expressed in this study, substitutions are underlined)

[0667]

[0668] >Hans-LanM-Cys (for immobilization)

[0669]

[0670] >Hans-LanM(R100K)-Cys (for immobilization)

[0671]

[0672] Figure 27 A non-exhaustive list of sequences that are likely to dimerize (in addition to residues at positions 72, 78, and 80 shown in the figure (corresponding to D85, E91, and D93 in the text figure), also includes the R100 equivalent residue, position 87 in the figure). These sequences are compared to sequences that are unlikely to dimerize, such as M. extorquens, and to some other specific sequences as described in the figure legends. This includes the following:

[0673] >LanM_011 (Hyphomicrobium, signal peptide removed)

[0674]

[0675] >LanM_012 (Flavobacterium, signal peptide removed)

[0676]

[0677] >Unclassified Hyphomicrobium (signal peptide removed; shown to have only 3 functional EF hands)

[0678]

[0679] A non-limiting set of variants is as follows:

[0680] Constructs with two Hans-LanM monomers connected by different amino acid linkers (Hans-linker constructs)

[0681] HansR100K-L1

[0682] HansR100K-L2

[0683] HansR100K-L3

[0684] HansR100K-L4

[0685] HansR100K-L5

[0686] Constructs in which Hans-LanM interface residues were mutagenized to alter dimerization affinity (all in the Hans-LanM background):

[0687] I43A、I43V

[0688] A44N, A44S, or A44T

[0689] I47A, I47V

[0690] M92L, M92A, or M92D

[0691] D93N, D93A, D93E

[0692] Combination of successful Hans-linker constructs with Hans-LanM interface residue variants

[0693] Mex-LanM variants that may alter the stoichiometry / affinity of metal binding:

[0694] A32D / A117K

[0695] A32D / A117R

[0696] I42L / N108D / I115L

[0697] G51A

[0698] A98G

[0699] A99G

[0700] V100G

[0701] A102G

[0702] N108D

[0703] A124G

[0704] A127G

[0705] Combination of the above

[0706] Key features of RE-dependent protein dimerization. Based on our results, we predict that the following features are sufficient for dimerization via the described mechanism: 1) LanM-like sequences. First, most possess four EF-hand motifs, although proteins with two or three EF-hands may also possess the second and third features described below (although such proteins may or may not dimerize via different mechanisms / interfaces). Second, with the exception of proteins with three EF-hands (one of which is damaged, thus leaving only a few carboxyl residues), the EF-hands are generally separated by 12-13 residues (the 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). To our knowledge, this spacing is unique among EF-hand proteins and can itself be used as a criterion for LanM. Third, at least one EF-hand contains a proline at the second position (in Mex-LanM, all four EF-hands have a P2 residue). Using the first two criteria and a sequence length of less than 200 residues, we identified 696 putative LanM.2. The following residue pattern, in sequence positions aligned with Hans-LanM D85, E91, D93, and R100, where D85, E91, and D93 correspond to positions 3, 9, and 11 of EF hand 3, while D93 and R100K appear to be unique residues in promoting dimerization, which is sensitive to the identity of the RE bound to the protein. The structure shows that other interactions are also involved in dimer stabilization, but the above residues are conserved among many LanM proteins and appear to be key to RE-sensitive dimerization.

[0707] Note that in principle, RE-dependent dimerization may not require the entire protein sequence, for example, not all residues in or near EF1 and EF4 in the primary sequence may be required. In other words, it is conceivable that shorter (more atom-economical) LanM-like proteins can also undergo RE-sensitive dimerization.

[0708] Furthermore, the selectivity trends between REEs, the steepness of the selectivity within REEs, and the overall strength of dimerization can be modulated (reduced or increased) by inducing mutations in interfacial polar and nonpolar residues as described herein.

[0709] Finally, other conceptually related dimerization mechanisms may also exist in other lanthanoid proteins (e.g., use of different dimerization interfaces through specific interactions between EF2 ligands), and indeed in other lanthanide-binding proteins.

[0710] Example 3

[0711] This example describes the use of the peptides / proteins of the present disclosure.

[0712] LanM_013 is of interest because EF2 resembles the EF hand of Hans-LanM (N1 residue and E9 residue, with Gly at position 4), while EF3 resembles the EF hand of Mex-LanM, and all EF hands contain a Pro residue. It is envisioned that the structure of EF2 will be very sensitive to the identity of the RE (like the Hans-LanM EF hand), but the protein is not expected to dimerize, thus demonstrating increased selectivity within the lanthanide series within a single LanM unit.

[0713] >LanM_013 (expression construct; signal peptide removed and N-terminal Met added, EF hand underlined):

[0714]

[0715] LanM_013 (full-length sequence of Methyloligella sp. GL2 with signal peptide underlined):

[0716]

[0717] The procedure used for the expression and purification of LanM_013 in E. coli was identical to that used for Mex-LanM, except that Ca(II) was not involved in the gel filtration step. Figure 56 The protein was purified to a high purity after the purification procedure. The volume of protein eluted from the S75 column was consistent with that of a monomer. The purification yielded 80 mg of LanM_013 (40 mg / L) from 2 L of culture medium. The protein has two Tyr residues; the extinction coefficient at 280 nm was 2980 M -1 cm (-1) .

[0718] Figures 57-59 The binding stoichiometry of LanM_013 was solved. Figure 57 For clarity, the absorbance peaks at 278 nm and 285 nm are tracked. Both peaks reach their maximum values ​​after the addition of 2.0 equivalents of La(III). Note that this assay specifically reports on the environment of tyrosine residues in proteins, which are located near EF arms 2 and 3. The total number of metal equivalents bound to proteins can be much higher.

[0719] Figure 58Figure 2 shows the fluorescence emission of Tyr residues in LanM_013 during titrations with La(III), Nd(III), and Dy(III). Upon addition of the first and second equivalents of La(III) and Nd(III), fluorescence initially decreases, then increases again after approximately 1.0 equivalent, similar to Mex-LanM, indicating that the protein can bind at least three equivalents of La(III) and Nd(III) under these conditions. Interestingly, this pattern was not observed for dysprosium, suggesting structural differences near one or both Tyr residues when LanM_013 binds to certain lanthanides. Figure 59 Competitive titrations with citrate using La(III)-bound LanM_013 (3 equivalents of La(III)) were again 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 was attributed to the weaker binding of the third equivalent of metal (see Figure 57 Even 100 mM citrate was unable to restore the tyrosine fluorescence to that of the apoprotein, indicating that the remaining two equivalents of metal (presumably bound to EF2 and EF3) were tightly bound, possibly more tightly than to Mex-LanM. In contrast, at approximately 20 mM citrate, the two most tightly bound equivalents of La(III) were desorbed by approximately 50% (see the Hans-LanM work). Figure 4 a). Slower equilibrium dynamics may also play a role in the LanM_013 system.

[0720] Figure 60 and 61 The stoichiometry and affinity of LanM_013 were solved. Figure 60 In , xylenol orange was used as a colorimetric competitor for the binding of La(III) and Dy(III). The results showed a slight increase in signal throughout the Dy(III) titration, with a significant increase in signal upon addition of 1 to 2 equivalents of metal, indicating relatively weak binding to Dy(III) compared to La(III). La(III) clearly showed about 2.0 equivalents of binding, which corresponds well with the other experiments described above. The third equivalent of La(III) binding clearly visible in the fluorescence experiments is presumably very weak; if it is weaker than about 1-10 μM, it would not be observed in the xylenol orange experiments. In Figure 61In this study, CD spectroscopy was used to assess the extent of conformational changes in LanM_013 upon lanthanide binding. Clear titration endpoints were observed under La(III) conditions (pH 5 and pH 7), while a wider range of responses was observed under Dy(III) conditions. This suggests that the affinity of at least one site in the Dy(III)-LanM_013 complex may be weaker than that of the La(III)-LanM_013 complex. Figure 62 In this study, CD spectroscopy was used to preliminarily estimate the apparent K of LanM_013 in response to some lanthanide elements. d The values ​​were 11 pM (La) and 96 pM (Dy). Both Hill coefficients were approximately 1.5.

[0721] Even in the absence of more detailed affinity measurements, these experiments allow for conclusions to be drawn. LanM_013 possesses several intriguing properties that could make it well-suited for LRE-only recovery or LRE / HRE separation. Metal binding to the two tightest binding sites (presumably EF2 / 3 based on other characterized LanMs) appears to induce structural changes immediately adjacent to Tyr (Y73), resulting in quenching of fluorescence emission from this residue in the presence of La(III) and Nd(III), but not in the presence of Dy(III). Additional biophysical data also indicate a significant difference in affinity between the heavy and light REs for at least one of these sites, although it remains to be assessed where within the lanthanide series the affinity "breakpoint" occurs. In this regard, the protein could exhibit effects similar to those of Hans-LanM, but achieved within a single polypeptide. The two tightest sites also appear to bind lanthanides more tightly than Mex-LanM.

[0722] In-depth crystallographic characterization of the Mex-LanM and Hans-LanM systems has enabled predictions about the origins of key properties of this novel protein, potentially extending their applicability to proteins with similar sequence features but not yet biochemically characterized. First, regarding the apparent structural differences between Dy-bound and La / Nd-bound LanM_013, the E9 residue (E44) in EF2 may have undergone a shift in coordination from bidentate (with the LRE) to monodentate (with the HRE), a shift that could be transmitted to EF3 in a manner that allows EF2 and EF3 to pair only when the LRE is present in each EF hand. The presence of a second glycine (G39) in EF2 could also shift the EF2-bound HRE into a conformation incompatible with EF3 interaction. The presence of an Asn residue as the first residue in EF2 could also play a role. Second, based on citrate data, the protein exhibits a significantly higher affinity for La(III) than Mex-LanM, a potential source of this higher affinity due to additional hydrogen bonding interactions unique to the LRE-bound form of the protein. Based on comparison with the Mex-LanM structure, residue E74 (Glu after EF3) is well positioned to hydrogen bond with K29 and / or K25. This interaction may also contribute to the different affinities of the LRE and HRE complexes for the protein.

[0723] Example 4

[0724] This example describes the use of the peptides / proteins of the present disclosure.

[0725] LanM_012 was used to sense Nd and Yb. The sensitization ability of Mex-LanM (T90W), Hans-LanM (R100K) and LanM_012 to the near-infrared (NIR) luminescence of Nd(III) and Yb(III) was tested. Figure 63 The data showed that all three sensors were able to sensitize Nd(III) and Yb(III) above pH 4.0. Only LanM_012 showed considerable intensity for Nd(III) at pH 3.0, and no sensor was able to sensitize Yb(III) at pH 3.0, which may be due to the lower binding affinity of LanM to the later REEs under low pH conditions. Overall, LanM_012 showed the best overall performance for both ions, especially for Yb(III), which showed very high luminescence intensity. Therefore, from all the work included in this article, LanM_012 is a particularly promising protein-based sensor for several individual REEs (Tb, Eu, Dy, Sm, Nd, and Yb) and can be used for in vitro REE studies and intracellular REE uptake studies.

[0726] Determine the dissociation constant (K) of LanM_013d At pH 5.0, the apparent dissociation constants of LanM_013 for three intermediate and later REEs with similar ionic radii were determined (Table 24 summarizes them). Figure 64 LanM_013 has a preference for lighter lanthanides such as Gd(III) over heavier lanthanides such as Dy(III) and Ho(III). In these titrations with single-class REEs, a rather skewed affinity trend emerges over a narrow atomic number range: a ~7-fold difference between Gd and Ho (only Tb and Dy fall between the two). The apparent K of LanM_013 for Gd is d Comparison of apparent K of Dy d is about 3 times tighter, and the apparent K of Dy d Comparison of Ho's apparent K d The binding of LanM_013 to Gd(III) is about 2.5 times tighter. In addition, the binding of light lanthanides is more cooperative than that of heavy lanthanides. LanM_013 exhibits a biphasic response to Gd(III), with a major cooperative phase and a minor non-cooperative phase ( Figure 64 A). If the measurement is performed at a higher metal concentration, it is possible to observe similar curves for Dy(III) and Ho(III) because this potential second phase is not considered in the fitting of Dy and Ho, and thus the apparent K d There may be slight differences from the numbers reported here. It is not clear whether the weaker phase is EF1, as this seems to be the case for other LanMs that have been studied. It is worth noting that these K d The slightly tighter results than previously reported may be due to the higher protein concentration used previously (20 μM vs. 10 μM), which slightly affected the free metal concentration in the titration and caused the calculated free metal concentration value to be slightly overestimated.

[0727] Table 24. LanM_013 and Gd III 、Dy III and Ho III Overview of fitting parameters for CD titration

[0728]

[0729] In order to quantitatively determine the selectivity of different lanthanomodulins within REEs, the single light (La-Dy, Figure 65 ) or heavy (Gd-Lu with Y, Figure 66) REE mixtures were subjected to equilibrium binding to immobilized protein, with each metal ion added at equimolar concentration (pH 5). Each solution was circulated on the LanM column for two hours. The distribution value (D) was determined by quantifying the distribution of REEs between the solid phase (i.e., LanM bound) and the aqueous phase. The D value was used to determine the separation factor by taking the ratio of the distribution values ​​between the two REEs of interest. For LanM_001, the highest selectivity was observed for the LREEs and MREEs Ce, Pr, Nd, Sm, and Eu, which formed a selectivity plateau showing minimal differences between the D values. LanM_002 and LanM_012 exhibited a narrower selectivity plateau centered on Pr and significantly enhanced selectivity between LREEs and HREEs. Compared to LanM_001 and LanM_002 / LanM_012, LanM_013 exhibited an intermediate selectivity trend, including preferential binding to Ce-Sm. Interestingly, LanM_013 exhibits the most selective shift towards Eu to Dy among all variants, producing separation factors close to 2 for Eu / Gd, Gd / Tb, and Tb / Dy. These variants are able to separate adjacent REEs (from Pr to Dy) more efficiently than LanM_001. Figure 66 ), the selectivity trend of Gd-Lu appears closer to LanM_001 than that of La-Dy data ( Figure 65 ). It is possible that the presence of LREEs enhances the selectivity among HREEs (perhaps through a mixed binding scheme).

[0730] Experimental Details: Batch experiments were performed to determine the distribution value and separation factor. LanM-immobilized microbeads were washed with deionized water. 5 mL of feed solution (3 mM total REEs, equimolar at pH 5) was added to 1 mL of microbeads and incubated for 2 hours. The equilibrium solution was collected and the REE concentration was determined as [M]adsorbed. The REEs were then desorbed with 4 mL of 0.1 M HCl, and the concentration was determined as [M]desorbed. The REE distribution value (D) between the LanM phase and the solution phase was calculated as:

[0731]

[0732] Among them, [M] LanM and [M] 液体 are the metal ion concentrations in the LanM solid phase and the unbound solution phase at equilibrium, respectively. To account for the free liquid absorbed within the agarose beads, the following corrections were made [M] 液体 =[M] 吸附 ; [M] LanM =([M] 解吸 *4-[M]吸附 *1) / 4. The separation factor (SF) is defined as follows:

[0733]

[0734] Among them D REE1 and D REE2 are the distribution values ​​of REE1 and REE2 respectively.

[0735] The binding capacity of Mex-LanM was improved by stabilizing metal binding to EF1 and EF4. Target sites for enhancing binding stoichiometry were EF1 and EF4. EF4 exhibits very weak metal binding, and at neutral pH, EF1's affinity is slightly lower than EF2 and EF3, but this affinity decreases further with decreasing pH. It is hypothesized that the increased volatility of EF1 is partly due to its pairing with EF4, which binds metal ions less tightly and is therefore more pH-sensitive. Furthermore, the low affinity of EF4 may be related to its preorganization (i.e., its adjacent helices are stabilized by metal binding to EF2 and EF3).

[0736] To achieve tighter binding of the REE to EF1 and EF4, three different site-directed mutagenesis strategies were employed. First, the amino acid sequence in EF2 and EF3 was replicated and the metal-binding loops in EF1 and EF4 were optimized. Notably, EF4 differs in that Asn occupies the first position (from this point on, subscript numbers indicate the position within the EF hand), rather than Asp1 as found in the other three EF hands. The non-binding side chain NH2 of Asn1 is unable to form a hydrogen bond with the backbone NH of Gly6, resulting in a different conformation of the loop. Perhaps for this reason, the 8th position in EF1 and EF4 is Ile, rather than Leu8 as in EF2 and EF3. Therefore, converting Asn1 in EF4 to Asp and Ile8 in EF1 and EF4 to Leu optimizes the lanthanide binding geometry. (Variant Group 1: N108D and I42L N108DI115L).

[0737] Furthermore, introducing a hydrogen bond between EF1 and EF4 could enhance the stability of the complex in a metal-dependent manner. Specifically, the crystal structure of Mex-LanM bound to Nd(III) revealed a hydrogen bond connecting position 3 (D56) of EF2 and position 10 (K93) of EF3. On the other hand, Ala residues occupy equivalent positions in EF1 and EF4. Therefore, mutating these Ala residues in a manner similar to that of EF2 and EF3 could establish a stable hydrogen bond (variant group 2: A32D / A117K and A32D / A117R).

[0738] It was hypothesized that the prestructured form of EF4 would result in less energy release during metal-induced conformational changes. Therefore, it was hypothesized that improved stoichiometry could be achieved by destabilizing the apo-binding loop. This would activate metal binding by EF4, thereby stabilizing the metal-bound conformation of EF1. In wt-Mex-LanM, EF2 and EF3 occupy the majority of the α-helical content in the protein. It was proposed that the α-helix (α2) between EF3 and EF4 is formed completely or largely upon REE binding to EF2 / 3; therefore, inhibiting complete α2 formation during metal chelation by EF2 and EF3 would allow for some stabilization during EF4 occupation. Therefore, three Ala residues located roughly in the middle of α2, which tend to stabilize the helix, were selectively mutated to more flexible Gly residues, which tend to destabilize the helix (variant group 3: A98G, A99G, and A102G).

[0739] The binding stoichiometry of the variants was investigated using xylenol orange (XO) competition assays and circular dichroism (CD) spectroscopy. The XO competition assay used a buffer of 20 mM acetic acid, 20 mM MES, 100 mM KCl, pH 6.1. CD spectroscopy was used to observe conformational changes in the variants during Nd titration. The CD buffer used was 20 mM acetic acid, 100 mM KCl, pH 5.0.

[0740] Variant Group 1: No variants exhibited improved binding stoichiometry. However, apoN108D was found to have significant α-helical content at pH 5.0, which was close to half of the maximal helicity ( Figure 67 B) When Asp is present at this position in EF4, it can form a hydrogen bond with the backbone NH of Gly at position 6, leading to the formation of α-helices around EF4. It is hypothesized that these α-helices are located between EF3 and EF4 and at the C-terminus of EF4. The I42L and I115L mutations appear to further stabilize the apo structure in conjunction with N108D, but the effect is minimal.

[0741] Variant Group 2: The A32D / A117R and A32D / A117K variants exhibited an improvement in binding stoichiometry of approximately 0.5 equivalents at pH 6.1 as determined by XO competition assay ( Figure 68 A). Although the same effect was not observed from the CD titration, it is important to note that the CD titration was performed at pH 5.0, so this could reflect the pH dependence of hydrogen bond strength ( Figure 68 B) The secondary structures of these variants are similar to wt LanM. Additional hydrogen bonds may contribute to the stabilization of the complex between EF1 and EF4, but this may require additional mutations (such as those that stabilize metal binding in EF4) to be fully effective.

[0742] Variant Group 3: No 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 69 A). However, all variants A to G exhibit reduced α-helicity in their apo states and exhibit less helicity than wt in the metal-bound state ( Figure 69 B) The reduction in apo-helicity of these variants suggests that α2 is partially ordered in the wt apoprotein or that it helps stabilize another part of the protein, such as the C-terminal α-helix following EF4.

[0743] "Combo" variant group: A variant group named "combo" was prepared, which combined mutations from variant groups 1 and 3 ( Figure 70 Among the options, A99G was selected because the CD titration curve appeared to show an improved second binding event ( Figure 71 B). Interestingly, the combination of the A99G and N108D mutations reduced the α-helicity of apo N108D back to near-wt levels. This observation suggests that conferring the ability of residue 108 to directly or indirectly hydrogen bond with the G113 backbone NH leads to interaction and stabilization with the α2 helix, which in turn interacts with the C-terminal helix. On the other hand, since the variant containing A99G exhibited neither a complete conformational change nor improved binding stoichiometry, it may not be a suitable position for mutation. The variant I42L A99G N108D I115L exhibited a slight improvement in Nd binding stoichiometry in the XO competition assay, but CD results indicated a decrease in binding stoichiometry at pH 5.0.

[0744] Therefore, the results suggest the following for stabilizing metal binding to EF1 and EF4. First, A32D, which carries a basic residue (most likely R) at residue 117, slightly stabilizes EF1, but is insufficient to confer stability at pH 5 and below. After further stabilizing EF4 metal binding, it is proposed that the A32D / A117R mutation could be combined with EF4-stabilizing mutations to fully stabilize EF1. Second, N108D is believed to be crucial for forming an optimized EF4-binding loop, but this must be combined with helix-destabilizing mutations in the apoprotein. Strategic introduction of Gly residues to affect this destabilization appears important, but A99G is likely not the right choice. Retaining the N108D mutation, an A to G mutation will be introduced in the region following EF4. Two Ala residues following Pro123 were selected: variants N108D A124G and N108D A127G will be expressed, purified, and subsequently characterized in a similar format. Variant N108D A102G can also be tested. Finally, since Hans-LanM and LanM_012 are characterized by an Asn at the first position of the EF hand (and different positions of Gly residues in the loop), partial loop sequences of these proteins can also be used for substitution into EF4.

[0745] >Mex-LanM-N108D / A124G (mutations are underlined)

[0746]

[0747] >Mex-LanM-N108D / A127G (mutations are underlined)

[0748]

[0749] >Mex-LanM-N108D / A102G (mutations are underlined)

[0750]

[0751] Although the present disclosure has been described with respect to one or more specific 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, the protein 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 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, in i) for the first EF-hand motif, the second EF-hand motif and the fourth EF-hand motif: Each X 1 independently D or N; Each X 2 independently any standard amino acid; Each X 3 independently D, N, or E; Each X 4 independently any standard amino acid; Each X 5 independently D, N, or E; Each X 6 independently any standard amino acid; Each X 7 independently any standard amino acid; Each X 8 are independently hydrophobic residues; Each X 9 independently D, E, or T; Each X 10 are independently hydrophobic residues; and Each X 11 independently any standard amino acid; ii) For the third EF-hand motif: X 1 It is N; X 2 is any standard amino acid; X 3 It is D; X 4 It is G or A; X 5 is D or N; X 6 is any standard amino acid; X 7 It is T or S; X 8 is a hydrophobic residue; X 9 It is E; X 10 is a hydrophobic residue; and X 11 is D; and iii) the EF-hand motifs are connected by a 12 or 13 amino acid residue linker, and each amino acid residue of the linker is a standard amino acid, except for the third EF-hand motif and the fourth EF-hand motif, which are connected by the following sequence: (X)5-R-(X)6, wherein each X is independently a standard amino acid, and at least one amino acid of any of the linkers is hydrophobic.

2. The protein according to claim 1, wherein the X of the first EF-hand motif, the second EF-hand motif and / or the fourth EF-hand motif is 7 are independently T or S.

3. The protein according to claim 1, wherein the X of the third EF-hand motif 4 It’s A.

4. The protein according to claim 1, wherein the X of the third EF-hand motif 8 It’s L.

5. The protein according to claim 1, wherein the X of the third EF-hand motif 10 It is L, I or M.

6. The protein of claim 1, wherein the protein comprises the following 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 KGRLTEKDWARANKDGDQTLEMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK(SEQ ID NO:2) or a protein with 70% identity to SEQ ID NO: 1 or SEQ ID NO:

2.

7. The protein of claim 1, wherein the protein comprises the following sequence: MLTGKEFLRKYNKDKDSTVEIVEAIDLGTKVFKAINPDKD K TLEAAETKGRLSDEDWAQFNKDGDKTLELDEWLIIVRKRFNDADANKDGKLTEAELDAPAGQQILLIAK(SEQ ID NO:7), 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 of claim 8, wherein the rare earth element is a heavy rare earth element.

11. The protein of claim 1 , wherein the protein comprises the following sequence: MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRRLTEKDWARANKDGDQTLEMDEWLKILX TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK(SEQ ID NO:44), wherein X is any standard amino acid residue except R.

12. The protein of claim 11, wherein the protein comprises the following sequence: MASGADALKALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWARANKDGDQTL EMDEWLKILKTRFKRADANK DGKLTAAELD SKAGQGVLVM IMK (SEQ ID NO: 4).

13. The protein of claim 1, wherein the protein has the following sequence: >Hans-LanM MKLSLKAGAA ITAFVFAASP VLAASGADAL KALNKDNDDS LEIAEVIHAGATTFTAINPDGDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILRTRFKRADANK DGKLTAAELD SKAGQGVLVMIMK(SEQ ID NO:1); >Hans-LanM MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRRLTEKDWARANKDGDQTLEMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK(SEQ ID NO:2); >Hans-LanM(R100K) MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRRLTEKDWARANKDGDQTLEMDEWLKILK TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK(SEQ ID NO:4); >Hans-LanM-Cys MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRRLTEKDWARANKDGDQTLEMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVMIMKGSGC(SEQ ID NO:5); >Hans-LanM(R100K)-Cys MASKADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWARANKDGDQTLEMDEWLKILK TRFKRADANK DGKLTAAELD SKAGQGVLVMIMKGSGC(SEQ ID NO:6); >LanM_012 MLTGKEFLRKYNKDKDSTVEIVEAIDLGTKVFKAINPDKD K TLEAAETKGRLSDEDWAQFNKDGDKTLELDEWLIIVRKRFNDADANKDGKLTEAELDAPAGQQLILLIAK(SEQ ID NO:7); >LanM_011 MGHHNCKAEMAYLNPDHDGTIDWREARRAAVRLFHKLDPDHDGTLDMKEVRGRVGILSFARFNPDRDGKLDKHEWLALVKHRFHRANPDKDGTIDCRELHSLAGRKLLRVLM(SEQ ID NO:14); >HansR100K-L1 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGGSGGSGGSGGSGGSGGSGGSAGSASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:16); >HansR100K-L2 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:17); >HansR100K-L3 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:18); >HansR100K-L4 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:19); >HansR100K-L5 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGSGGSGAEAAAKEAAAKAGGSGGSAEAAAKEAAAKAGSGGSGASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:20); >Hans-LanM-I43A MASGADALKALNKDNDDSLEAAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:26); >Hans-LanM-I43V MASGADALKALNKDNDDSLEVAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:27); >Hans-LanM-A44N MASGADALKALNKDNDDSLEINEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:28); >Hans-LanM-A44S MASGADALKALNKDNDDSLISEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:29); >Hans-LanM-A44T MASGADALKALNKDNDDSLITEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:30); >Hans-LanM-I47A MASGADALKALNKDNDDSLEIAEVAHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:31); >Hans-LanM-I47V MASGADALKALNKDNDDSLEIAEVVHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:32); >Hans-LanM-M92L MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLELDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:33); >Hans-LanM-M92A MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEADEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:34); >Hans-LanM-M92D MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEDDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:35); >Hans-LanM-D93A MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMAEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:36); >Hans-LanM-D93N MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMNEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:37); >HansR100K-L1 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGGSGGSGGSGGSGGSGGSGGSAGSASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:16); >HansR100K-L2 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:17); >HansR100K-L3 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:18); >HansR100K-L4 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:19); >HansR100K-L5 MASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMKGSGGSGAEAAAKEAAAKAGGSGGSAEAAAKEAAAKAGSGGSGASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKGRLTEKDWARANKDGDQTLEMDEWLKILKTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK(SEQ ID NO:20); or >Hans-LanM(3E9Q) MASGADAL KALNKDNDDS LQIAEVIHAG ATTFTAINPD GDTTLQSGET KGRLTEKDWA RANKDGDQTL QMDEWLKILR TRFKRADANK DGKLTAAELDSKAGQGVLVM IMK(SEQID NO:38).

14. The protein according to claim 1, wherein the protein has the following sequence: MKLSLKAGAA ITAFVFAASP VLAASGADAL KALNKDNDDS LEIAEVIHAGATTFTAINPDGDTTLESGET KGRLTEKDWA RANKDGDQTL EMDEWLKILRTRFKRADANK DGKLTAAELD SKAGQGVLVMIMK(SEQ ID NO:1); MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGETKGRLTEKDWA RANKDGDQTL EMDEWLKILR TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK(SEQ IDNO:2); or MASGADAL KALNKDNDDS LEIAEVIHAG ATTFTAINPD GDTTLESGET KGRLTEKDWARANKDGDQTL EMDEWLKILK TRFKRADANK DGKLTAAELD SKAGQGVLVM IMK(SEQ ID NO:4).

15. A protein with enhanced REE / REE selectivity, the protein 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 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, in i) for the first EF-hand motif and the fourth EF-hand motif: Each X 1 independently D or N; Each X 2 independently any standard amino acid; Each X 3 independently D, N, or E; Each X 4 independently any standard amino acid; Each X 5 independently D, N, or E; Each X 6 independently any standard amino acid; Each X 7 independently any standard amino acid; Each X 8 are independently hydrophobic residues; Each X 9 independently D, E, or T; Each X 10 are independently hydrophobic residues; and Each X 11 independently any standard amino acid; ii) For the second EF-hand motif: X 1 It is N; X 2 is any standard amino acid; X 3 It is D; X 4 is any standard amino acid; X 5 It is D; X 6 is any standard amino acid; X 7 It is T or S; X 8 is a hydrophobic residue; X 9 It is E; X 10 is any standard amino acid; and X 11 is any standard amino acid; and iii) For the third EF-hand motif: X 1 It is D; X 2 is any standard amino acid; X 3 It is D; X 4 It is D; X 5 It is D; X 6 It is G; X 7 It is T or S; X 8 is a hydrophobic residue; X 9 It is D; X 10 is any standard amino acid; and X 11 is any standard amino acid; iv) at least one X of the second EF-hand motif and the third EF-hand motif 2 is P; and v) the EF-hand motifs are connected by a 12 or 13 amino acid residue linker, wherein each amino acid of the linker is a standard amino acid and at least one amino acid of any of the linkers is hydrophobic.

16. The protein of claim 15, wherein the protein has the following sequence: >Mex-LanM-G51A MAPTTTTKVDIAAFDPDKDGTIDLKEALAAASAAFDKLDPDKDGTLDAKELKGRVSEAD LKKLDPDNDGTLDKKEYLAAVEAQFKAANPDNDGTIDARELASPAGSALVNLIR(SEQ ID NO:21); >Mex-LanM-A98G MAPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEAD LKKLDPDNDGTLDKKEYLGAVEAQFKAANPDNDGTIDARELASPAGSALVNLIR (SEQ ID NO: 22); >Mex-LanM-A99G MAPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEADLKKLDPDNDGTLDKKEYLAGVEAQFKAANPDNDGTIDARELASPAGSALVNLIR (SEQ ID NO: 23); >Mex-LanM-V100G MAPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEADLKKLDPDNDGTLDKKEYLAAGEAQFKAANPDNDGTIDARELASPAGSALVNLIR (SEQ ID NO: 24); >Mex-LanM-A102G MAPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEADLKKLDPDNDGTLDKKEYLAAVEGQFKAANPDNDGTIDARELASPAGSALVNLIR(SEQ ID NO:25); >LanM_013 MGKAADAIQALDPDKDGTIDLNEAKAGAKAVFEKINPDGDGTLEVKELKGRLTKKELDAADPDNDGTLDMQEYEAVVTKQFELANPDNDGTVDEKELKTKEGKKLLKLIY(SEQ ID NO:8); >Mex-LanM-A32D / A117R MAPTTTTKVDIDAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAANPDNDGTIDRRELASPAGSALVNLIR(SEQ ID NO:12); >LanM_013: MAAILTIAGAVTVAAGGAAFAGKAADAIQALDPDKDGTIDLNEAKAGAKAVFEKINPDGDGTLEVKELKGRLTKKELDAADPDNDGTLDMQEYEAVVTKQFELANPDNDGTVDEKELKTKEGKKLLKLIY(SEQ ID NO:9); >Mex-LanM-A32D / A117K MAPTTTTKVDIDAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEADLKKLDPDNDGTLDKKEYLAAVEAQFKAANPDNDGTIDKRELASPAGSALVNLIR(SEQ ID NO:11); >Mex-LanM-I42L / N108D / I115L MAPTTTTKVDIAAFDPDKDGTLDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEAD LKKLDPDNDGTLDKKEYLAAVEAQFKAADPDNDGTLDARELASPAGSALVNLIR(SEQ ID NO:13); >Mex-LanM-N108D / A124G MAPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEAD LKKLDPDNDGTLDKKEYLAAVEAQFKAADPDNDGTIDARELASPGGSALVNLIR(SEQ ID NO:39); >Mex-LanM-N108D / A124G MAPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEAD LKKLDPDNDGTLDKKEYLAAVEAQFKAADPDNDGTIDARELASPAGSGLVNLIR(SEQ ID NO:40); >Mex-LanM-N108D / A102G MAPTTTTKVDIAAFDPDKDGTIDLKEALAAGSAAFDKLDPDKDGTLDAKELKGRVSEAD LKKLDPDNDGTLDKKEYLAAVEGQFKAADPDNDGTIDARELASPAGSALVNLIR(SEQ ID NO:41); >Unclassified Hyphomicrobium MGHRSAKAHPSCPALNAIDPDGDGAMTLGEAKRAAIKTFMKLNKDGDITLELDELGGRM SAAAFAQADLIKGRGISLGEYLIEVRRRFKWANPDKDHTIECDELHSKYGRLLARLLK (SEQ ID NO: 15); >Mex-LanM-N108D OR >Methyloligella halotolerans MADAEISDTMKVVDPDMDNALTLEEAQAAGAKVFKKLNTDDDNTLEADELKGRVSERQLKKADPDDDGSLDMAEYEALIKKRFEAANPDGDDTIESDELETKKGKKLLELIQE (SEQ ID NO: 10).

17. The protein according to claim 15, wherein the X of the second EF-hand motif and / or the third EF-hand motif 8 It is L, I, M or V.

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

19. The device of claim 18, wherein the device is a filter, a membrane, a sensor, a handheld detector, a microplate reader, a fluorometer, a biosensor, or an online monitor.

20. A kit comprising: The protein according to claim 1 or claim 15; or A device comprising a protein according to claim 1 or claim 15.

21. A method of separating rare earth elements, the method comprising contacting the protein according to claim 1 with a sample comprising a rare earth element, wherein the rare earth element binds to one or more proteins according to claim 1, and removing the proteins from the sample.

22. The method of claim 21, wherein the sample is drinking water, wastewater, groundwater, ash ponds, aqueous extracts of contaminated soil, drainage water, leachate, solid waste (e.g. electronic waste), or aqueous extracts or leachates of ores or tailings, or solid samples.

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

24. The method of claim 21, wherein the method further comprises detecting and quantifying the one or more rare earth elements.

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

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

27. A method of determining the presence of a light rare earth element 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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