Artificial Diiron Enzymes with a De Novo Designed Four‐Helix Bundle Structure

Abstract

A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal-binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four-helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

Design and engineering of artificial oxygen-activating metalloenzymes

“…120-122 The overall results obtained on DF family demonstrated that the DF structure represents an excellent scaffold for hosting different activities. The earliest developed models have contributed to our understanding of the principles governing protein folding, stabilization, as well as metal coordination and substrate binding.…”

Identification of Protein Functional Regions

“…A possible way to construct artificial evolution pathways with such characteristics is the inverse protein folding, better known as protein design . Protein design consists in identifying sequences specific for a given backbone structure that at equilibrium would spontaneously adopt the target conformation.…”

De Novo Design of Tetranuclear Transition Metal Clusters Stabilized by Hydrogen-Bonded Networks in Helical Bundles

“…Our groups have designed the DF ( Due Ferri ) series of di-metal carboxylate bridged clusters stabilized helical bundles, 55,56 originally designed as mimics of diiron proteins. A number of versions of these proteins were prepared in which the four-helix bundle was assembled from four helical peptides, two helix-loop-helix peptides or a recombinantly expressed single-chain protein.…”