Metal‐Directed Self‐Assembly in a Beta‐Sheet Miniprotein Scaffold

Natural proteins exploit oligomerization, buried hydrophobic pockets, and metal coordination to drive catalysis and signaling. Miniproteins, as simplified protein‐like scaffolds, provide a tractable system for studying sequence–structure–function relationships while leveraging chemical synthesis to explore chemical space beyond biological constraints. Here, we introduce V14C, a miniprotein scaffold derived from the 27‐residue “Foldon” domain of T4‐fibritin, which oligomerizes into a trimer by selectively binding to zinc and cadmium within its hydrophobic pocket. Circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and electrospray ionization‐mass spectrometry (ESI‐MS), demonstrate that V14C binds to Zn 2+ with a dissociation constant of 8.5 µM. Since V14C is a weakly trimerizing Foldon variant, strong thiophilic heavy metal ions like Hg 2+ and Cd 2+ can impose their preferred coordination geometry on its scaffold. Consequently, V14C‐Hg 2+ forms a dimer, while V14C‐Cd 2+ assembles into a native‐like trimer. This work demonstrates the potential of identifying and rationally engineering stably folding natural domains to obtain miniprotein scaffolds with new properties. Such top‐down engineered systems can be used to explore new chemical environments, expand the toolkit for metallo‐protein studies and promote the rational design of functional biomolecular architectures for potential applications in sensing and catalysis.