Cobalt-Binding Peptide 1

Usage

Cobalt-binding peptide 1 (CBP1) is a human designed peptide from the N-terminal region of human serum albumin (HSA).It can bind Co(Ⅱ) specificaly. We will display it with Pichia pastoris through cell-surface display systems.

Biology

In a study, researchers designed wAlb12(CBP1) bound to Co(II) and evaluated its properties, intensity, and the binding site for use as a chelating agent. After binding with Co(II), the structure of wAlb12 changed and the chemical shifts were represented. Additionally, using LC-MS, we confirmed the increased absorbance intensity and molecular weight. The Co(II) self-assembled with wAlb12 showed strong stability at neutral or basic pH and different temperatures. The binding of the peptide to Co(II) resulted in increased stability against proteolytic digestion. Furthermore, we confirmed that there was no cytotoxicity in rat and human cell lines in vitro. The H3A analog, which substituted histidine with alanine, and acetylated wAlb12 did not bind to Co(II). Thus, the N-terminus of the α-amino group and 3-histidine are essential sites that interact with Co(II) ^[1].

Simulation

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^[2-6], in order to predict the binding ability. In the following video: white is H, gray is C, blue is N, red is O, flesh color is Co, purple is Li, light green is Cl, dark green is Ni.
(1) Sequence
Here we perform machine learning quantum chemistry calculations for the sequence CBP1: DAHKSEVAHRFK. Firstly, the geometric structure of the polypeptide chain was optimized, and the folded configuration was obtained, as shown below.
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Vid. 1 Structure of CBP

(2) Structural analysis of single ion binding
A Co²⁺ atom is added to the result in (1) to simulate the binding of the folded peptide to the ion. Through structural optimization, we get the following results:
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Vid. 2 Structure of CBP binding 1 Co²⁺

It can be seen that the coordination atoms with Co²⁺ are mainly carbonyl O and amine N atoms on the polypeptide chain. N=C double bonds may also participate in coordination.
(3) The combination of multiple ions
We designed this metal-binding peptide in the hope that they could trap multiple metal ions and increase efficiency. In order to analyze the binding of multiple Co²⁺ ions, four Co²⁺ ions were added to the molecular model, and four Cl^- ions were added to balance their charge, with a total charge of +4. Through structural optimization, we get the following results:
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Vid. 3 Structure of CBP binding 4 Co²⁺s

It can be seen that the N and carbonyl O atoms of higher amines participate in the coordination, and the counterion Cl^- also participates in the coordination, and the electrostatic interaction between the counterion and the H atom on the polypeptide also has a certain effect on the stability of the system. At the same time, it can be seen from observation that there are still more N and carbonyl O atoms of higher amines as coordination active sites, which can bind more Co²⁺ ions.

Experiments

1.We used Pichia Pastoris GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the CBP1 metal-binding peptide gene as the target gene, we obtained the corresponding surface display plasmid.

Fig. 1 Electrophoretic map of plasmids containing MBPs gene.

2.After constructing the plasmid, we introduced it into Escherichia coli for amplification. After amplification, the plasmids were extracted and purified, and sent for sequencing. After obtaining the correct sequencing results, the plasmid was transformed into Pichia pastoris by means of electrical stimulation. Finally, by colony PCR, we determined that the plasmid was successfully introduced into the yeast.

Fig. 2 Electrophoretic map of engineered yeast after colony PCR.

3.After obtaining the engineered yeast, we designed some experimental schemes to qualitatively test their adsorption effect on target metal ions. In order to test the adsorption effect, the engineered yeast and the prepared single metal solution were mixed according to a certain proportion, removed and centrifuged after 2 hours, and the supernatant and precipitation were stored respectively. We chose to use a graphite furnace to detect the concentration of metal ions in the supernatant. By calculating the ratio of the reduced metal concentration in the supernatant to the original added metal concentration, we obtained the adsorption rate of the engineered strain on the target metal ions, and made a comparison to select the strain with better adsorption effect.

Fig. 3 The adsorption rate of CBP1-pir and CBP2-pir for Co²⁺ within 2 hours.

Reference

[1]Cho Y, Mirzapour-Kouhdasht A, Yun H, et al. Development of Cobalt-Binding Peptide Chelate from Human Serum Albumin: Cobalt-Binding Properties and Stability[J]. Int J Mol Sci, 2022,23(2).
[2]Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/
[3] Q. Sun, et al. J. Chem. Phys. 2020, 153, 024109
[4] Q. Sun, et al. WIREs Comput. Mol. Sci. 2018, 8, e1340
[5] Q. Sun, J. Comp. Chem. 2015, 36, 1664
[6] L.-P. Wang, C. C. Song, J. Chem. Phys. 2016, 144, 214108

Sequence and Features

Assembly Compatibility: