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.