Part:BBa_K5261001

TFD-S, a monomeric protein for rare earth adsorption

The TIM-FD-Single (TFD-S) protein adsorbs lanthanide metal ions in solution and quantitatively detects lanthanide binding using a tryptophan-enhanced ‘antenna effect’ luminescence mechanism.

TFD-S is a monomeric protein based on TFD-EE, which is a homodimeric protein fusion. It combines TIM barrel folding and ferredoxin folding with a large protein inner cavity, and its unique structure can provide lanthanide ion coordination using glutamate residues.

Different lanthanide metals have different fluorescence emission wavelengths, but due to the low extinction coefficient of lanthanide metals, they cannot be directly excited, and the indole ring on the surrounding tryptophan absorbs external energy and then transfers it to the lanthanide metal ions, which are then excited to produce fluorescence.

Usage and Biology

TFD-S is a monomeric protein based on TFD-EE [1], which is a de novo designed homodimeric protein fusion. It combines TIM barrel folding and ferredoxin folding with a large protein inner cavity, and its unique structure can provide lanthanide ion coordination using glutamate residues. TFD-EE has a high affinity for lanthanide ions (equilibrium dissociation constant KD ≈ 10−18 M), significantly higher than the natural lanthanide binding protein, lanmodulin (KD ≈ 10−12 M), and high tolerance to thermal and chemical cosolvents [1]. So we want to use it for the biomining of rare earth elements. However, if the homodimer protein TFD-EE is to be applied in the field of biosorption, such as constructing whole-cell biosorbents through yeast surface display, the self-assembly of protein monomers may be limited by surface display systems. Therefore, we first fused the homologous dimer protein gene into a single monomer protein gene (Figure 1. B), named TFD-S, through a head-to-tail junction, and its corresponding metal binding sites are E6, E52, E175, E221.

Figure 1. The metal binding site of TFD-S (four glutamate residues), and the tryptophan antenna residues

pET-28a (+)-TFD-S Plasmid Construction

We sent the TFD-S gene sequence to Azenta for gene synthesis and cloned it into the pET-28a (+) plasmid vector. We received E. coli containing the pET-28a (+)-TFD-S plasmid (Figure 2. A). After sequencing verification, the successful insertion of the TFD-S gene was confirmed by sequence comparison (Figure 2. B, C).

Figure 2. A. pET-28a (+)-TFD-S plasmid B. TFD-S gene sequence C. The sequencing results showed that the TFD-S gene was successfully inserted into the pET-28a (+) plasmid

Induced expression, purification, and SDS-PAGE process of TFD-S in Escherichia coli BL21 (DE3)

Preparation of reagents

Lysis Buffer: 300 mM NaCl, 20 mM Tris, 20 mM Imidazole, 5 mM PMSF, 100 ug/mL Lysozyme, 10 μg/mL DNase (pH = 8.0)

Wash Buffer: 25 mM HEPES, 300 mM NaCl, 20 mM Imidazole (pH 7.5)

Elution Buffer: 25 mM HEPES, 300 mM NaCl, 300 mM Imidazole (pH 7.5)

Shaking Flask Cultivations

1. 10 µL of Escherichia coli seed solution stored in glycerol tube was collected on LB solid medium containing 50 µg/mL Kan, strewn, inverted plate, and incubated in a constant temperature incubator at 37℃ overnight.
2. Single colonies were picked from the plate into a test tube containing 5 mL of LB liquid medium, and 5 µL Kan at a concentration of 100 mg/mL was added and incubated overnight at 37℃ on a shaker at 220 rpm.
3. The inoculum was transferred to 100 mL LB liquid medium containing 50 µg/mL Kan, and incubated at 37℃ for 4 h until OD600 reached about 0.6-0.9, then 50 µL 0.5 M IPTG was added. Expression was induced for 16 h at 18℃.
4. After cultivation, the bacterial solution was collected and centrifuged at 4000 rpm for 30 min. The supernatant was discarded, and the bacteria were resuspended in 10 mL Lysis Buffer and placed in an ice water bath for ultrasonic lysis. The parameters of ultrasonic cell lysis were 520 W, 3 s ultrasonic work, 7 s interval, and 150 cycles. The cytoclastic fluid was centrifuged at 4000 rpm for 20 min, and the supernatant containing the target protein was collected.

Purification

pET28a (+)-TFD-S has a 6×His tag added to the C terminus, which can specifically bind to Ni ions, so the Ni-NTA affinity column was used for protein purification experiments.

1. The Ni-NTA packing column was cleaned with 5 times the column volume of deionized water and then equilibrated with 5 times the column volume of Lysis Buffer.
2. The supernatant obtained by cell lysis was further filtered by 0.22 µm filter membrane to avoid contaminating the packed column, and the sample was repeated 5 times to fully combine the protein with the Ni column.
3. Wash Buffer with 10 times the column volume to remove impurities and wash out the residual miscellaneous proteins in the column.
4. Finally, the target protein was eluted with 3 times the column volume Elution Buffer and the elution solution was collected.
5. The column material was eluted with 5x column volume Elution Buffer and cleaned with 5x column volume deionized water, and the Ni-NTA affinity column was preserved with 20% alcohol.
6. Pour the protein eluent into a Millipore ultrafiltration tube with a molecular weight cut-off of 10 kDa, centrifuge at 4000 rpm for 20 min, add deionized water to the original volume, then centrifuge, repeat the operation for 3 times, absorb the protein concentrate into a 1.5 mL EP tube, and store at 4℃.

SDS-PAGE Validation

1. Protein sample preparation: 40 µL protein solution was sucked and mixed with 10 µL SDS-PAGE protein loading buffer, heated at 99℃ for 10 min, and then loaded after cooling.
2. Electrophoresis: MOPS buffer was added to the electrophoresis tank, and 5 µL protein marker and 10 µL protein sample were added to the loading well, respectively. The voltage was set at 160 V, and the electrophoresis time was 1 h.
3. Staining: After the end of electrophoresis, the protein glue was removed, the dye Coomassie Brilliant blue R250 was added to immerse it, and the staining was shaken for 30 min.
4. Decolorization: Dip the dyed protein glue into water and shake it overnight to decolorize until clear protein bands are seen.
5. Imaging: Image acquisition was performed in a gel imager.

Finally, we expressed and purified TFD-S protein in Escherichia coli BL21(DE3), which was verified by SDS-PAGE, indicating that the TFD-S protein was successfully obtained (theoretical band 37.49kDa) (Figure 3).

Figure 3. SDS-PAGE and Coomassie bright blue staining of TFD-S (Note: TFD-S-1 and TFD-S-2 are two parallel samples. Lane 1: Initial supernatant, Lane 2: precipitation after centrifugation, Lane 3: buffer elute, Lane 4: Dilute imidazole elute, Lane 5: concentrated imidazole elute, Lane 6: protein concentrate obtained after ultrafiltration)

Computer Visualization and Simulations

AlphaFold3 Prediction

We fused this dimer protein into a monomeric protein, TFD-S, and gave the sequence to AlphaFold3 for prediction, finding that its spatial structure was almost identical to that of the TFD-EE protein. The characterization experiments showed that its performance was similar to that of TFD-EE dimer (Figure 4). Subsequently, site-specific mutation will be carried out on the basis of TFD-S.

Figure 4. Combination of TFD-S and Tb(III)

Molecular Dynamics Simulation Analysis of TFD-S

The crystal structure of TFD-S is predicted by AlplaFold3, which is a TFD single-chain protein with a terbium ion. MD simulation was performed using CHARMM36 protein Force Field 45 in the GROMACS2022.3 package 46. The force constant of the harmonic bias potential is 1000 kJ∙mol-1∙nm-2. Set the 10 Å cuboid box where the edge is widest from the protein, use the TIP3P water model in it, and add sodium ions to the solution to maintain the neutrality of the system. Eleven TFD-S-X(III) ( X(III) means Tb(III) or La(III) or Lu(III) ) systems were simulated at 300 K. To begin with, the system is minimized by 50,000 steps using the steepest descent algorithm. In the pre-balancing phase, the system is gradually heated to 300 K at 1 atm using a 200ps NVT set and 200ps NPT set. A 50ns production run followed to collect the balanced configuration for each 2ps interval. A speed scaling thermostat 49 with a time constant equal to 0.1ps was used to keep the temperature constant throughout the simulation. To maintain the pressure, the Berendsen pressure coupler was used in the NPT pre-balance run and the Parrinello-Rahman pressure coupler was used in the production run, with the pressure time constant and isothermal compression rate set at 2ps and 4.5×10-5 bar-1, respectively. In the whole simulation process, the integral time step of the motion equation is 2fs. The cut-off value for the non-bonding interaction is 12Å. The particle grid Ewald algorithm 53 is used to calculate long range electrostatic interactions. [2] The results showed that the average distance between the 8 active oxygen atoms of TFD-S and terbium ions was 2.41 Å (Figure 5).

Figure 5. Schematic diagram of binding of eight active oxygen atoms to terbium ions in TFD-S. The Tb(III) ion is shown as green sphere, and coordination bonds are shown as dashed lines. The numbers near the coordination bonds represent the distances between the atoms, with the unit of Å.

Figure 6. The RMSD curve of TFD-S protein

Figure 7. Molecular dynamics simulation structure of TFD-S protein

Protein Characterization

Tryptophan has been installed near the rare earth binding site in the internal cavity of the TFD-S protein as an antenna group (W10 and W179), which serves as a site-specific sensitizer for lanthanide luminescence. When 280 nm wavelength exciting light is given, the indole ring on tryptophan absorbs the energy of light and transfers it to terbium ion. Through the energy transfer mechanism, terbium ions are excited to a high-energy state. In the excited state, terbium ion releases photons through a radiative transition to produce characteristic luminescence at 545 nm wavelength. [3] This can be used to detect rare earth bonding.

Binding ability of TFD-S to different lanthanide metal ions

Firstly, the binding of TFD-S and Tb(III) was characterized by the terbium luminescence mechanism sensitized by antenna effect. The 10 μM TbCl3 and 5 μM TFD proteins were pre-incubated in Tris-HCl buffer pH 7.5 for 1 hour, then added into 96-well plates. The excitation wavelength was set at 280nm, the detection emission wavelength ranged from 520nm to 570nm, and the step length was 5nm using a time-resolved fluorescence mode (TRF). Detection of terbium emission enhanced by tryptophan. Subsequently, to detect the adsorption capacity of proteins for different rare earth ions, Tb(III) in the incubated proteins was replaced with other lanthanide ions of different concentrations (except radioactive element Pm(III)), and the luminescence intensity was detected every 30 min, and the decay curve of the Tb(III) fluorescence value enhanced by tryptophan was measured over time (Figure 8).

The interference factors of fluorescence value measured in this experiment mainly come from the spontaneous fluorescence of rare earth ion (trace), the spontaneous fluorescence of protein (trace), and the fluctuation of fluorescence value with time due to the dynamic factors in the binding equilibrium process after terbium ion binds to protein. The fluorescence intensity caused by the first two interfering factors is minimal. To eliminate the interference of time fluctuation factors, we set the blank control group as: 180 μL incubation mother solution and 20 μL buffer solution, that is, no replacement ions were added, so that the attenuation of fluorescence value only came from the binding and replacement process of TFD-S. The data of the experimental group were corrected by subtracting the control group's data at the corresponding time. At 0 h, the control group with terbium ion binding at all the protein adsorption sites had the strongest fluorescence intensity, so the other data were all negative.

Figure 8. Tb(III) fluorescence decay curves of TFD-S-Tb(III) after substitution by different rare earth ions.

Additionally, the equilibrium dissociation constant KD can usually reflect the affinity of protein and ligand, and the two are inversely proportional. To characterize the affinity of TFD-S to different rare earths, we selected the relatively stable-binding ions Pr(III)/Nd(III)/Sm(III)/Eu(III) for analysis and mapping, and obtained the attenuation curve of the characteristic luminescence intensity of Tb3+ over time (Figure 9, left), as well as the fitting curve of the replacement ion concentration with the equilibrium fluorescence intensity of Tb3+ (Figure 9, right).

Figure 9. The binding of other lanthanides, such as Pr(III), Nd(III), Sm (III), and Eu(III), was measured in displacement titrations using Tb(III)-bound TFD-S. The left figure showed the attenuation curve of the characteristic luminescence intensity of Tb3+ with time, and the right figure showed the fitting curve of the replacement ion concentration and the equilibrium fluorescence intensity.

When the fluorescence value of Tb(Ⅲ) decays by half, it can be considered that the number of protein adsorption sites occupied by the replacement ion X(Ⅲ) and Tb(Ⅲ) is equal, and they have the same binding ability to the protein, that is, KD(Tb3+)/KD(X3+)=c(Tb3+)/c1/2(X3+), X represents the other rare earth element. Therefore, in the fitting curve of the replacement ion concentration and equilibrium fluorescence intensity, c1/2(X3+) at half attenuation of A545 was extracted, and KD(Tb3+)/KD(X3+) was obtained. Shane J. Caldwell et al. titrated the pre-incubated TbCl3 and EDTA mixture with metal-free TFD protein, and obtained an equilibrium dissociation constant KD(Tb3+/EDTA) = 1.6×10−18 M [1], which can be used as a reference. The final results are shown in the following table.

Table 1. Fitting results

Measurement of binding capacity of lanthanide metal elements by TFD-S under different pH conditions

Rare earth ore bioleach is usually acidic, so we need to further examine the difference in the binding ability of TFD protein to lanthanide metals under acidic conditions. We selected Tb(III) ion for adsorption experiment. Prepare 100 mM Tris-HCl buffers at pH 6.5, 7, 7.5, 8, and 8.5 using Tris-HCl and sodium hydroxide solutions at pH 6.8. For lower pH buffers, as the commonly used citric acid/phosphoric acid buffers will react with rare earth ions, we use 1 M hydrochloric acid to prepare solutions at pH 2, 3, 4, 5, 5.5, and 6. Each well was added in accordance with the incubation system of 200 μL, and the test was started immediately with an enzyme-labeled instrument (the parameters were the same as those of the adsorption displacement experiment) at a time interval of 15 min. The curves of the adsorption reaction of TFD-S with Tb(III) at different pH were obtained. For TFD-S (Figure 10), the emission intensity of terbium in the experimental pH range all increased with the increase of time, indicating that TFD-S had adsorption capacity for Tb(III) in the pH range of 5.0~8.5. The most suitable environment is pH=6.5, when terbium luminous intensity is the highest, the adsorption capacity of protein to Tb(III) is the strongest.

Figure 10. Adsorption of Tb (III) by TFD-S in the pH range of 5.0-8.5

Yeast surface display of TFD-S

To further characterize the usability of TFD-S in biological systems, we presented the TFD-S protein on the yeast surface using an α-lectin-based yeast surface display system. We cloned the TFD-S gene into the C-terminal position of the AGA2 anchoring protein gene on the pYD1 plasmid vector to achieve the fusion expression of the AGA2 and TFD genes, thereby displaying the lanthanide adsorbed protein TFD-S on the cell surface.

Figure 11. A. The pYD1-TFD plasmid. B. AGA2-TFD gene fusion expression on pYD1-TFD plasmid. C. The sequencing results showed successful insertion of the TFD gene into the pYD1 plasmid.

In addition, we used AlphaFold3 to predict the spatial structure of the Aga2-TFD fusion protein and found that the spatial structure of the TFD can still be well maintained (Figure 12).

Figure 12. Spatial structure of the Aga2-TFD fusion protein predicted by AlphaFold3.

Quantitative detection of rare earth ions adsorbed by surface-displaying strains of bacteria

Next we further tested the ability of engineered yeast to adsorb rare earth ions. Add 100 μM of TbCl3 to the EBY100 (pYD1-TFD) cell culture medium. After shaking incubation for one day, centrifuge to precipitate the cells and measure the concentration of free Tb(III) in the supernatant. Cell culture medium without TbCl3 was used as a blank control. Three parallel experiments were done for each data point. Due to the high price of ICP-MS or ICP-OES, here we adopted the Arsenazo III dye-based assay method, which can be used to roughly determine the rare earth ion content in the solution, see our Protocols page for more details. The absorbance values at 650 nm can reflect the rare earth ion content within a certain range.

After the adsorption process, the TbCl3 content in the solution decreased significantly, which was manifested in the obvious color depth difference of the Arsenazo III – REE complex formed before and after adsorption (Figure 3-6 A), which was also reflected by the value of A650. However, due to the limitation of the Arsenazo III assay and the extremely low concentration of Tb(Ⅲ) ions in the solution after adsorption, we could not quantitatively detect the exact concentration of rare earth ions by drawing a standard curve. However, we could roughly estimate it by the "two-point interval method" according to the value of A650: For the solution after adsorption, three parallel data points were measured in a microplate reader, and the A650 obtained was between that of 2 μM and 10 μM TbCl3 standard solution (Figure 3-6 B), so it could be preliminarily judged that c(Tb3+) in the solution after the adsorption was also in this range. Since we initially added c(Tb3+) = 100 μM (before adsorption), it could be considered that our surface display strain had a strong adsorption capacity for rare earth ions.

Figure 13. A. The absorbance at 650 nm of the solution before and after biosorption was determined by Arsenazo III assay, and obvious color depth difference was observed. B. A650 value of standard solution of TbCl3 from 0 to 150 μM.

References

[1]Caldwell, Shane J., et al. Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion. Proceedings of the National Academy of Sciences, 2020, 117(48): 30362-30369.

[2]Tutorials and Webinars — GROMACS webpage https://www.gromacs.org documentation

[3] Martin, Langdon J., and Barbara Imperiali. The best and the brightest: Exploiting tryptophan-sensitized Tb3+ luminescence to engineer lanthanide-binding tags. Peptide Libraries: Methods and Protocols. 2015: 201-220.

Sequence and Features

Assembly Compatibility:

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