An Efficient, Step-Economical Strategy for the Design of Functional Metalloproteins

The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. While numerous strategies have been used to create new metalloproteins, preexisting knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (Metal Active Sites by Covalent Tethering or MASCoT) whereby folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naïve protein-protein interfaces. Metalloproteins generated with this strategy uniformly bind a wide array of first-row transition metal ions (Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for Mn(II) to 50 fM for Cu(II)). MASCoT readily affords coordinatively unsaturated metal centers, including a five-His coordinated non-heme Fe site, and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands like nitric oxide to the interfacial metal center.