Cobalt-Binding Peptide 2

Usage

Cobalt-binding peptide 2 (CBP2) is a cobalt-binding peptide from the Ph.D.™-C7C Phage Display Peptide Library (New England Biolabs GmbH, Frankfurt/Main, Germany). It can bind Co(Ⅱ) specificaly. We will display it with Pichia pastoris through cell-surface display systems.

Biology

In the study, a cobalt-binding one with the peptide motif CTQMLGQLCGGG was isolated. The cobalt-binding peptide showed better affinities of about factor 20 for both elements, the more moderately binding nickel peptide showed selectivity for its target ion under these particular experimental conditions^[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 CBP2: CTQMLGQLCGGG. 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 CBP2.

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

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Vid. 2 Structure of CBP2 binding 1 Co²⁺.

It can be seen that the coordination atoms with Co²⁺ are mainly S atoms on the polypeptide chain, and O and N atoms also participate in the 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, three Co²⁺ ions were added to the molecular model, and four Cl^- ions were added to balance their charge, with a total charge of +2. Through structural optimization, we get the following results:

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Vid. 3 Structure of CBP2 binding 3 Co²⁺.

It can be seen that the counterion Cl^- is also involved in the coordination, and the 3 Co²⁺ are wrapped by the polypeptide to form clusters through Cl bridging bonds, and the counterion also plays a certain role in stabilizing the system.

Experiments

1.We used Pichia Pastoris GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the CBP2 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

[1Matys, S., Morawietz, L., Lederer, F., & Pollmann, K. (2022). Characterization of the Binding Behavior of Specific Cobalt and Nickel Ion-Binding Peptides Identified by Phage Surface Display. Separations, 9(11), 354. doi: 10.3390/separations9110354]
[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] Sun, Q., Berkelbach, T., Blunt, N., Booth, G., Guo, S., Li, Z.,... Chan, G. (2018). PYSCF: the Python-based simulations of chemistry framework. WILEY INTERDISCIPLINARY REVIEWS-COMPUTATIONAL MOLECULAR SCIENCE, 8(1). doi: 10.1002/wcms.1340
[4] Sun, Q. (2015). Libcint: An efficient general integral library for Gaussian basis functions. JOURNAL OF COMPUTATIONAL CHEMISTRY, 36(22), 1664-1671. doi: 10.1002/jcc.23981
[5] Wang, L., & Song, C. (2022). Geometry optimization made simple with explicit translation and rotation coordinates (vol 144, 214108, 2016). JOURNAL OF CHEMICAL PHYSICS, 157(1). doi: 10.1063/5.0102029
[6]Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/

Sequence and Features

Assembly Compatibility: