Understanding the molecular mechanisms underlying metalloenzyme catalysis has been a longstanding goal in the field of bioinorganic chemistry. To this end, rational mutagenesis and directed evolution have been ubiquitous, "top-down” strategies used to identify functional residues and redesign active sites of naturally occurring metalloproteins. A compelling alternative to these methods is de novo design of a metalloprotein, whereby metal-binding sites are grafted onto a small and highly tunable protein scaffold. A de novo designed metalloprotein that trimerizes in the presence of five first row transition metals (FeII, CoII, NiII, CuII, ZnII) is reported. The metal binding promiscuity was characterized in solution using analytical ultracentrifugation, while metal binding affinities were estimated using competitive binding titrations. Crystal structures revealed a suite of highly conserved, non-covalent interactions, making this trimer a promising scaffold for the design of a mononuclear, open coordination site mimicking (His)3-FeII active sites. We report on our efforts to build a supramolecular (His)3-FeII site through Rosetta-based redesign of the trimeric interface and medium throughput screening of Rosetta-generated variants.

De novo design of a mononuclear, unsaturated FeII active site in a supramolecular protein trimer

2019 Fall National Meeting

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