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We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis–Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter K m and a 7-fold decrease in k cat . These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch. 

Abstract De novodesign provides an attractive approach, which allows one to test and refine the principles guiding metalloproteins in defining the geometry and reactivity of their metal ion cofactors. Although impressive progress has been made in designing proteins that bind transition metal ions including iron–sulfur clusters, the design of tetranuclear clusters with oxygen‐rich environments remains in its infancy. In previous work, we described the design of homotetrameric four‐helix bundles that bind tetra‐Zn²⁺clusters. The crystal structures of the helical proteins were in good agreement with the overall design, and the metal‐binding and conformational properties of the helical bundles in solution were consistent with the crystal structures. However, the correspondingapo‐proteins were not fully folded in solution. In this work, we design three peptides, based on the crystal structure of the original bundles. One of the peptides forms tetramers in aqueous solution in the absence of metal ions as assessed by CD and NMR. It also binds Zn²⁺in the intended stoichiometry. These studies strongly suggest that the desired structure has been achieved in theapostate, providing evidence that the peptide is able to actively impart the designed geometry to the metal cluster.