Breaking symmetry: Metal-guided assembly of heterotrimeric coiled coils

This chapter presents a method for preparing heterotrimeric parallel three-stranded coiled coils (3SCCs) using Pb(II)-templated design to generate dissymmetric metal-binding sites. The systematic variation of hydrophobic core residues, specifically leucine and alanine, at positions adjacent to the Pb(II)(Cys)₃ metal-binding site allows us to exploit the balance between hydrophobic packing and internal solvation of 3SCC to favor the formation of defined and pure heterotrimeric assemblies (e.g., A₂B or AB₂, where A and B are different strands). The goal of this approach is to relax the intrinsic C₃ symmetry of the 3SCC scaffold to enable the installation of a divalent metal-binding site at the C-terminus, allowing for the construction of dissymmetric coordination environments. The individual strands are short de novo designed peptides, each composed of fewer than 40 amino acids and synthesized via solid-phase peptide synthesis. ²⁰⁷Pb NMR spectroscopy is used to confirm heterotrimer formation, while Co(II) UV-Vis electronic absorption spectroscopy probes the local coordination environment at the C-terminus metal binding site. This strategy provides a versatile platform for designing de novo protein scaffolds that incorporate functional metal-binding sites, capturing the inherent dissymmetry characteristic of metal binding sites in natural metalloenzymes.