# Design of Zn‐Binding Peptide(s) from Protein Fragments

## TL;DR

Researchers designed a zinc-binding peptide using fragments from known protein structures and confirmed its binding properties experimentally.

## Contribution

A novel computational protocol was developed to design zinc-binding peptides using structural constraints and validated experimentally.

## Key findings

- A peptide with nanomolar zinc-binding affinity was synthesized and confirmed via ITC.
- The zinc coordination involves both cysteines, as shown by proton release during binding.
- NMR and CD spectroscopy confirmed structural similarity to computational predictions.

## Abstract

We designed a minimalistic zinc(II)‐binding peptide featuring the Cys2His2 zinc‐finger motif. To this aim, several tens of thousands of (His/Cys)‐X
n
‐(His/Cys) protein fragments (n=2–20) were first extracted from the 3D protein structures deposited in Protein Data Bank (PDB). Based on geometrical constraints positioning two Cys (C) and two His (H) side chains at the vertices of a tetrahedron, approximately 22 000 sequences of the (H/C)‐X
i
‐(H/C)‐X
j
‐(H/C)‐X
k
‐(H/C) type, satisfying N
metal–binding H=N
metal‐binding C=2, were processed. Several other criteria, such as the secondary structure content and predicted fold stability, were then used to select the best candidates. To prove the viability of the computational design experimentally, three peptides were synthesized and subjected to isothermal calorimetry (ITC) measurements to determine the binding constants with Zn2+, including the entropy and enthalpy terms. For the strongest Zn2+ ions binding peptide, P1, the dissociation constant was shown to be in the nanomolar range (KD
=~220 nM; corresponding to ΔG
bind=−9.1 kcal mol−1). In addition, ITC showed that the [P1 : Zn2+] complex forms in 1 : 1 stoichiometry and two protons are released upon binding, which suggests that the zinc coordination involves both cysteines. NMR experiments also indicated that the structure of the [P1 : Zn2+] complex might be quite similar to the computationally predicted one. In summary, our proof‐of‐principle study highlights the usefulness of our computational protocol for designing novel metal‐binding peptides.

We have designed a short zinc‐binding peptide by taking fragments from proteins with known structure. We have measured the binding affinity with isothermal calorimetry and the structure of the peptide using CD and NMR spectroscopies. These results were computationally supported by molecular dynamics and density functional theory calculations.