Part:BBa_K5166003

Nickel-Binding Peptide 4

Introduction

Nickel serves as a key component in ternary lithium batteries, and its similarity to cobalt complicates their separation and recovery. Designing innovative bioreactors capable of efficiently recovering and separating nickel from cobalt is of significant importance.

Inspiration

Peptides were selected as the core component tool for binding nickel ions due to their favorable biocompatibility, potential of rational design and self-assembly, and structural diversity.However, in their monomeric form, metal ion-binding peptides possess limited binding sites, whereas self-assembling polypeptides can form distinct binding sites. Consequently, we sought to enhance the binding efficiency of metal ion-binding peptides through a self-assembly strategy, exemplified in the case of N4.
We planned to display the peptide on yeast and express it at high throughput so that the peptide could self-assemble on the bacterial surface in situ. A large number of metal ions will be enveloped in the assembly process, and the assembly formed at the end of the assembly can also provide a large number of metal ion binding sites, which will make the adsorption of nickel more efficient.

Biology

NBP4 is selected from Phage Display Library Screening: The Ph.D.^™-C7C Phage Display Peptide Library (New England Biolabs GmbH, Frankfurt/Main, Germany). A study has shown that NBP4 selectively binds to nickel in TBS with NaCl at higher ionic strength.The sequence of N4 was CNAKHHPRCGGG, along with related properties such as GRAVY and isoelectric point (pI), is as follows:

Fig. 1 Basic parameters of peptides a)Plot of pH versus Net charge b)Monomer structure and table of associated parameters.

Here, peptides were characterized by isothermal titration calorimetry (ITC) with respect to their binding capacity for the respective target ion and binding experiments were performed on other ions.
We used a Nano-ITC for our experiments using a standard protocol. The measuring cell contained 350 µL buffer with 0.2 mM peptide. The syringe was filled with 2 mM LiCl, NiCl₂, MnCl₂, or CoCl₂ solution in the same buffer. The injections were performed in 20 single steps of 2.5 µL each. The stirring speed was 350 rpm. Using Launch NanoAnalyze, perform data evaluation.

Fig. 2 Isothermal titration calorimetry results of NBP4 with different mental ions.

The isothermal titration experiments revealed the cyclic nickel-binding peptide with the motif CNAKHHPRCGGG showed complexation with Ni²⁺ ions with an affinity of KD=-5.53x10^-5M(Figure 2). However, no interaction was detected between the nickel-binding peptide and Li⁺, Mn²⁺, and even Co²⁺. The nickel-specific peptide binds only selectively to nickel, and this powerful function will be of great importance in nickel-cobalt separation.

Modeling

We used the MLatom calculation program on the XACS platform to perform structural calculations of the binding between metal ion binding peptides and metal ions, to predict the binding ability. The results are as follows:
In the following videos, white represents H, gray represents C, blue represents N, red represents O, incarnadine represents Co, purple represents Li, light green represents Cl, and dark green represents Ni.
(1) Sequence
Here we performed a quantum chemical calculations based on the machine-learning for the sequence NBP4:CNAKHHPRCGGG. First, the geometric structure of the polypeptide chain was optimized to obtain the folded configuration, as shown below.

Video 1 the structure of NBP4.

(2) Structural analysis of single ion binding
We simulated the binding of a single Ni ion to the folded polypeptide chain. Through structural optimization, we obtained the following results:

Video 2 the structure of NBP4 combined with a single Ni²⁺.

It can be seen that the atoms coordinated with Ni²⁺ are mainly O and N atoms on the polypeptide chain. Ni²⁺ ions were encapsulated.
(3) Binding of multiple ions
We designed this metal binding peptide, hoping that they can capture multiple metal ions and improve efficiency. To analyze the binding of multiple Ni²⁺ ions, we added four Ni²⁺ ions to the molecular model. To balance its charge, we added four Cl^- ions with a total charge of +4. Through structural optimization, we obtained the following results:

Video 3 the structure of NBP4 combined with four Ni²⁺.

It can be seen that the N and carbonyl O atoms of the higher amine participate in the coordination, the sulfhydryl S atom participates in the coordination, the counterion Cl^- participates in the coordination, and the counterion also interacts with the carbon skeleton, which can adjust the charge distribution of the polypeptide chain as a whole, adjust the structure of the polypeptide, and stabilize the system.

Simultaneously, we employed AlphaFold3 to predict the structures of a series of metal-binding peptides, including N4. Among all the peptides, we observed that the 32-polymers of N4 exhibited an antiparallel double-layered β-sheet architecture, characterized by extensibility and translational repeatability. The laterally extending hydrogen bonds, in conjunction with the hydrophobic interactions between layers, contribute significantly to the structural stability. Therefore, we have sound reasons to postulate that the 32-polymers structure of N4 predicted by AlphaFold3 demonstrates its potential for self-assembly.

Fig. 3 AlphaFold3 structure prediction for N4 a)With the increase of the number of multimers, it can be seen that it has the characteristics of extensibility b)Local close-up of the 32-polymers.

Experiments

Given our design and the complexity of the cellular environment, we have divided the overall experimental validation into three distinct segments: yeast surface display of N4, in vitro self-assembly of chemically synthesized polypeptides, and co-incubation of self-assembled polypeptides with cells.

Yeast surface display of N4
1.We first designed and synthesized a plasmid containing NBP4 and introduced it into Pichia pastories.(The design of this surface display plasmid was based on the composition part we declared under the code BBa_K5166032)
We first designed the upstream and downstream primers according to the sequence of the short peptide NBP4 and amplified the plasmid containing NBP4 by PCR reaction. It was preliminarily verified by agarose gel electrophoresis that NBP4 had been introduced into the plasmid.

Fig. 4 Electrophoretic pattern of the plasmid.

After that, we introduced the plasmid containing NBP4 into E. coli, screened the E. coli that introduced the target gene plasmid in LB solid medium containing bleomycin through the bleomycin resistance marker contained in the plasmid, and extracted the plasmid for testing.
Then, we electroporated the correctly sequenced plasmid (containing NBP4) into Pichia pastoris, screened out the plasmid introduced Pichia pastoris in the solid medium containing bleomycin, and further verified the plasmid introduction by colony PCR.

Fig. 5 Electrophoresis patterns verified by PCR of colonies.

Finally, we preserved Pichia pastoris with the correct plasmids for subsequent adsorption experiments.
2.The nickel ion-binding peptide NBP4 was expressed on the surface of Picpastoris by surface display system, and the bacteria were placed in metal solution. AAS was used to measure the changes of metal ions in the solution to detect the adsorption ability of NBP4 to different metal ions. The control experiments with other metal ion-binding peptides showed that Pichia pastoris containing NBP4 plasmid had a better adsorption effect on nickel ions.

Fig. 6 Adsorption rate of AAS element detection.

In vitro self-assembly of chemically synthesized polypeptides
Based on the N4 sequence and the fundamental principles of polypeptide self-assembly, we assume two possible pathways for N4 self-assembly: low-temperature-induced hydrogen bond formation, with hydrogen bonds serving as the primary driving force for self-assembly; and high-temperature-promoted hydrophobic interactions, with hydrophobic interactions as the primary driving force. Additionally, considering the specific binding affinity of the imidazole group on the histidine R-group towards metal ions, we contemplate using a catalytic amount of metal ions to induce N4 self-assembly. Therefore, we have devised a series of experiments to validate the self-assembly of N4.

Fig. 7 a)AFM topography b)Characterization of the height of nanoparticle.

Through AFM characterization experiments, we can clearly observe from the images that under the conditions of 37°C and with catalytic amounts of Ni, N4 forms a large number of uniform spherical assemblies after 48 hours of assembly. In contrast, when the reaction is conducted at 4°C, there are almost no discernible assemblies formed by N4. Similarly, if heating is applied without the addition of metal ions, the assemblies formed by N4 are irregular, appearing as "tadpole-like" structures with small tails rather than spherical shapes.
Simultaneously , we observed that the height of the assembly was different under different conditions, and the trend was the same as the change of size.In a word, the assemblies obtained by Ni induction are large, round and abundant.
Among various metal ions tested for induction, Ni exhibits the most effective induction, which independently verifies the specific binding ability of N4 towards Ni.

Hypothysis of the self-assembly
Based on the existing self-assembly experimental data and theoretical calculations, we propose a possible self-assembly hypothesis: at an ambient temperature of 37 degrees Celsius, the elevated temperature promotes molecular thermal motion and hydrophobic interactions, thereby increasing the likelihood of peptide collisions and their proximity in aqueous solutions. Due to their specific binding affinity for nickel, the introduced Ni ions in the environment will induce the proximity and binding of two randomly moving peptides. Subsequently, this forms a nucleation point, attracting more peptides towards it. Under the influence of hydrophobic interactions, hydrogen bonding, and other forces, the N4 peptide gradually adopts a stable secondary structure and ultimately exhibits a nanospherical morphology.

Fig. 8 N4 self-assembling Schematic diagram.

In summary, our experimental validation confirms that N4 possesses the capability of self-assembly and that the structure of self-assembly can be optimized under our controlled conditions.

Co-incubation of self-assembled polypeptides with cells
After verifying the successful assembly of peptides into nanospheres, we plan to co-incubate the peptide assemblies with cells to validate cellular loading of the assemblies. We have designed two sets of control experiments. The first set involves co-incubating the assemblies alone with nickel chloride solution and observing them under SEM-EDS to verify whether the binding of the assemblies to nickel metal can be successfully detected by SEM-EDS. The second set of experiments entails co-incubating successfully assembled peptides with cells, followed by co-incubation with a metal ion solution. Meanwhile, a control group is configured, where dissolved peptides are immediately incubated with cells, allowing them to interact with cells in their monomeric form, before co-incubation with the metal ion solution. Additionally, we have established a blank control, where a solution without dissolved peptides is co-incubated with cells and then directly with the metal ion solution.

Fig. 9 a)scheme of the combination of the assemblies b)EDS analysis of electronic images c)EDS spot scanning carbon element content distribution image d)EDS point scanning images of oxygen content distribution e)EDS spot scanning image of nickel content distribution f)EDS surface scanning nickel content layered image g) Total spectrum of element distribution map of self-assembled N4 yeast incubated with Ni²⁺ for 3h in blank control group h) Total spectrum of element distribution map of self-assembled N4 yeast incubated with Ni²⁺ for 3h i) Analysis of 3h EDS of N4-added yeast incubated with Ni²⁺ (experimental group) j) 3h EDS analysis of yeast that has been added with N4 incubated with Ni²⁺ (taking no liquid at the edge as control group) k) Analysis of 6h EDS of N4-added yeast incubated with Ni²⁺ (experimental group) l) 3h EDS analysis of yeast that has been added with N4 incubated with Ni²⁺ (no liquid at the edge was taken as control group).

From the analysis of the results presented in the figures, it is evident that while the morphology of the spheres is difficult to directly observe under SEM due to their small size, elemental analysis by electron spectroscopy reveals a higher concentration of Ni in regions where peptides are present. This finding provides reasonable grounds to conclude that these are peptide assemblies enriched with Ni. In the second set of experiments, we clearly observe that after co-incubation of monomers with cells, there is almost no detection of high concentrations of Ni on the cell surface However, in the experiments where assemblies were co-incubated with cells, a pronounced difference in Ni content was observed between the yeast surface and the substrate. Given that each peptide monomer contains two histidine residues, and assuming each set of four histidines can complex one Ni ion, theoretically, each mole of peptide can bind half a mole of Ni. Based on this rough calculation, we estimate the Ni content in the peptide self-assemblies to be approximately 2.5%, which closely aligns with the experimentally measured value of approximately 2.3%. This substantial agreement validates our theoretical estimation.
In this part of the experiment, we have successfully demonstrated that the assemblies can bind to cells, and that these cell-bound assemblies are capable of adsorbing significant amounts of Ni ions. Furthermore, we have verified the indispensable advantage of assemblies over monomers, which we attribute to the additional binding sites provided by the assemblies. These extra sites not only facilitate the adsorption of more metal ions but also enable greater numbers of peptide segments to bind to cells.

Discussion

Fig. 10 Schematic design of the “fishing net” system.

In this segment of our work, we have innovatively constructed a biometal recovery system based on the yeast surface display of Ni-binding peptides facilitated by polypeptide self-assembly. The mechanism of this system is analogous to 'fishing.' The engineered yeast functions as a fishing boat navigating the ocean, while the secreted peptides assemble into “ nets”. The metal ions in the solution represent the “fish”, and once a large number of 'fish' are captured, the assembled complex is adsorbed by the 'fishing boat.' By collecting the 'fishing boats,' we can efficiently harvest a substantial quantity of metal ions.
Our independent validation process has confirmed three key aspects: the successful display of metal ion-binding peptides on the yeast surface, the ability of these peptides to self-assemble under certain conditions, and the successful binding of the assembled metal ion-binding peptides to the yeast surface, exhibiting robust metal ion-binding capabilities. This achievement not only lays a solid foundation for subsequent research endeavors but also stimulates thought-provoking insights within related fields. By demonstrating the feasibility and effectiveness of this novel system, we have opened up new avenues for exploring the potential applications of polypeptide self-assembly in biometal recovery and surface engineering.

Reference

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[2]Sun, Q., Zhang, X., Banerjee, S., Bao, P., Barbry, M., Blunt, N.,... Chan, G. (2020). Recent developments in the PySCF program package. JOURNAL OF CHEMICAL PHYSICS, 153(2). doi: 10.1063/5.0006074
[3]Raymond, D., & Nilsson, B. (2018). Multicomponent peptide assemblies. CHEMICAL SOCIETY REVIEWS, 47(10), 3659-3720. doi: 10.1039/c8cs00115d
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Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]