A De Novo Designed Metalloprotein Displays Variable Thermal Stability and Binding Stoichiometry with Transition Metal Ions

TL;DR

A newly designed protein folds when bound to specific metal ions and shows different stability and metal binding depending on the metal type.

Contribution

A de novo protein that folds upon binding transition metals and exhibits variable thermal stability and metal stoichiometry is presented.

Findings

The protein folds into an α-helical bundle when bound to Co(II), Ni(II), Cu(II), or Zn(II).

Cu(II) forms the least stable complex, and higher metal concentrations reduce secondary structure.

Metal binding affects protein stability through coordination geometry and backbone distortion.

Abstract

Metal‐binding selectivity in natural proteins is determined by multiple factors such as the protein's structure, metal concentration within cellular compartments, and the presence of metallochaperones. The in vitro selectivity of proteins for transition metal ions is largely governed by the Irving–Williams series, which states protein‐metal complex stability follows the order Co(II) < Ni(II) < Cu(II) > Zn(II). A de novo protein has been designed that folds in the presence of certain transition metal ions into a monomeric α‐helical bundle, with the least stable protein‐metal complex being formed with Cu(II). Moreover, when increasing the metal concentration of Cu(II) or Zn(II), more metal ions are incorporated into the protein accompanied by a concurrent decrease in the amount of secondary structure. One reason may be that there is a balance between stability conferred by the coordination of the metal ion(s) and stability conferred by hydrophobic packing of the α‐helical bundle. Metals may therefore adopt distorted coordination geometries, or binding of multiple ions may cause distortion of the protein backbone, leading to compromised folding of the protein scaffold, or variable thermal stabilities of the metalloprotein complexes. This protein scaffold therefore contributes to the deciphering of design rules for metal selectivity in proteins.

A de novo designed single‐chain coiled coil protein is unfolded in apo‐state but folds up upon addition of divalent transition metal ions Co(II), Ni(II), Cu(II), and Zn(II). The resulting metal‐bound complexes exhibit distinct thermal stability and metal stoichiometry, making this protein a promising starting point for the design of fully metal‐selective de novo proteins.© 2025 WILEY‐VCH GmbH

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