Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a non-catalytic protein scaffold, we recently generated a novel RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely novel structure with dramatically enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.

A major goal in metalloprotein design is to build protein scaffolds from scratch that allow precise control over metal coordination. A particular challenge in this regard is the construction of allosteric systems in which metal coordination equilibria are coupled to other chemical events that take place elsewhere in the protein scaffold. We previously developed a metal-templated self-assembly strategy (MeTIR) to build supramolecular protein complexes with tailorable interfaces from monomeric building blocks. Here, using this strategy, we have incorporated multiple disulfide bonds into the interfaces of a Zn-templated cytochrome cb562 assembly in order to create mechanical strain on the quaternary structural level. Structural and biophysical analyses indicate that this strain leads to an allosteric system in which Zn2+ binding and dissociation is remotely coupled to the formation and breakage of a disulfide bond over a distance of >14 Å. The breakage of this strained bond upon Zn2+ dissociation occurs in the absence of any reductants, apparently through a hydrolytic mechanism that generates a sulfenic acid/thiol pair.

CONSPECTUS: Nature puts to use only a small fraction of metal ions in the periodic table. Yet, when incorporated into protein scaffolds, this limited set of metal ions carry out innumerable cellular functions and execute essential biochemical transformations such as photochemical H 2 O oxidation, O 2 or CO 2 reduction, and N 2 fixation, highlighting the outsized importance of metalloproteins in biology. Not surprisingly, elucidating the intricate interplay between metal ions and protein structures has been the focus of extensive structural and mechanistic scrutiny over the last several decades. As a result of such top-down efforts, we have gained a reasonably detailed understanding of how metal ions shape protein structures and how protein structures in turn influence metal reactivity. It is fair to say that we now have some idea−and in some cases, a good idea−about how most known metalloproteins function and we possess enough insight to quickly assess the modus operandi of newly discovered ones. However, translating this knowledge into an ability to construct functional metalloproteins from scratch represents a challenge at a whole different level: it is one thing to know how an automobile works; it is another to build one. In our quest to build new metalloproteins, we have taken an original approach in which folded, monomeric proteins are used as ligands or synthons for building supramolecular complexes through metal-mediated self-assembly (MDPSA, Metal-Directed Protein Self-Assembly). The interfaces in the resulting protein superstructures are subsequently tailored with covalent, noncovalent, or additional metal-coordination interactions for stabilization and incorporation of new functionalities (MeTIR, Metal Templated Interface Redesign). In an earlier Account, we had described the proof-of-principle studies for MDPSA and MeTIR, using a four-helix bundle, heme protein cytochrome cb 562 (cyt cb 562 ), as a model building block. By the end of those studies, we were able to demonstrate that a tetrameric, Zn-directed cyt cb 562 complex (Zn 4 :M1 4 ) could be stabilized through computationally prescribed noncovalent interactions inserted into the nascent protein−protein interfaces. In this Account, we first describe the rationale and motivation for our particular metalloprotein engineering strategy and a brief summary of our earlier work. We then describe the next steps in the "evolution" of bioinorganic complexity on the Zn 4 :M1 4 scaffold, namely, (a) the generation of a self-standing protein assembly that can stably and selectively bind metal ions, (b) the creation of reactive metal centers within the protein assembly, and (c) the coupling of metal coordination and reactivity to external stimuli through allosteric effects.

Owing to their central roles in cellular signaling, construction, and biochemistry, protein-protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive non-covalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, Metal-Directed Protein Self-Assembly (MDPSA) and Metal-Templated Interface Redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution.

Despite significant progress in protein design, the construction of protein assemblies that display complex functions (e.g., catalysis or allostery) remains a significant challenge. We recently reported the de novo construction of an allosteric supramolecular protein assembly (Zn-R1) in which the dissociation and binding of Zn ions were coupled over a distance of 15 Å to the selective hydrolytic breakage and formation of a single disulfide bond. Zn-R1 was constructed by Zn-templated assembly of a monomeric protein (R1, a derivative of cytochrome cb) into a tetramer, followed by progressive incorporation of noncovalent and disulfide bonding interactions into the protein-protein interfaces to create a strained quaternary architecture. The interfacial strain thus built allowed mechanical coupling between the binding/dissociation of Zn and formation/hydrolysis of a single disulfide bond (C38-C38) out of a possible six. While the earlier study provided structural evidence for the two end-states of allosteric coupling, the energetic basis for allosteric coupling and the minimal structural requirements for building this allosteric system were not understood. Toward this end, we have characterized the structures and Zn-binding properties of two related protein constructs (R1 and R1) which also possess C38-C38 disulfide bonds. In addition, we have carried out extensive molecular dynamics simulations ofR1 to understand the energetic basis for the selective cleavage of the C38-C38 disulfide bond upon Zn dissociation. Our analyses reveal that the local interfacial environment around the C38-C38 bond is key to its selective cleavage, but this cleavage is only possible within the context of a stable quaternary architecture which enables structural coupling between Zn coordination and the protein-protein interfaces.

Inspired by the remarkable sophistication and complexity of natural metalloproteins, the field of protein design and engineering has traditionally sought to understand and recapitulate the design principles that underlie the interplay between metals and protein scaffolds. Yet, some recent efforts in the field demonstrate that it is possible to create new metalloproteins with structural, functional and physico-chemical properties that transcend evolutionary boundaries. This essay aims to highlight some of these efforts and draw attention to the ever-expanding scope of bioinorganic chemistry and its new connections to synthetic biology, biotechnology, supramolecular chemistry and materials engineering.

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