DF-Click: a modular approach to the de novo design of metalloprotein catalysts

Metal cofactors play a crucial role in regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals adopting only few ligands and slightly different geometries. Thus, understanding how the fine-tuning of the scaffolds hosting them imparts the wide spectrum of reactivity is of crucial interest both in the fields of structural biology and bioinorganic chemistry. We focused on the four-helix bundle as scaffold for metal binding sites in the context of protein de novo design, to obtain basic biochemical components for biosensing or catalysis. In this contest, we previously developed, through an iterative design process , C2 symmetric four helical bundles (DFs) that contain dicarboxylate bridged di-nuclear metal centers. Previous attempts to design asymmetrical DFs gave successful results . Here, an alternative synthetic approach to generate new DF analogues by means of copper catalyzed azidealkyne cycloaddition (CuAAC), also known as "Click Chemistry" [6], is implemented. Our design strategy led to a helix-loop-helix heterodimer, obtained through side chain chemical ligation, affording the DF-Click series.