Design and Construction of Functional Supramolecular Metalloprotein Assemblies

Abstract

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.

Mimochrome, a metalloporphyrin‐based catalytic Swiss knife†

“…Other groups, including ours, tried to practice with the "protein ligand," in an effort to test our understanding of the fundamental mechanisms that drive heme cofactor modulation [33]. This was and still is performed either by engineering natural scaffolds or by constructing entirely new-to-nature proteins [93][94][95][96][97][98][99][100][101][102][103][104][105]. These approaches demonstrated to be powerful not only to reproduce and/or optimize the biological functions of heme-proteins but also to construct artificial metalloenzymes that catalyze reaction with unknown natural counterparts [106,107].…”

Stimulus-responsive Self-Assembly of Protein-Based Fractals by Computational Design

“…Self-assembly of engineered proteins 23 provides a general framework for the controllable and bottom-up fabrication of novel biomaterials with chosen supramolecular topologies but these approaches have, thus far, been applied to the design of integer (two or three)-dimensional ordered patterns such as layers, lattices, and polyhedra 24–30 . While external triggers such as metal ions and redox conditions have been used to trigger synthetic protein and peptide assemblies 20,21,31–34 , phosphorylation – a common biological stimulus used for dynamic control over protein function – has yet to be utilized for controlling protein assembly formation.…”

“…S1). The resulting propagatable placements were evaluated using RosettaMatch 31 for geometrically feasible fusion to the SH2 domain and phosphopeptide with the C-terminal AtzC and N-terminal of AtzA, respectively (Fig. S2A,B).…”

Clickable artificial heme‐peroxidases for the development of functional nanomaterials

“…The research field now appears to have limitless potential, as protein scaffolds can be shaped, optimized, or repurposed to obtain artificial catalysts highly competent toward specific functions. Indeed, several outstanding examples demonstrate that it is possible to obtain artificial metalloenzymes behaving as oxydases, oxygenases, and peroxidases [1,2,[8][9][10][11][12][13][14], hydrolases [1,2,[15][16][17], and hydrogenases [1,18,19,]. The astonishing Biotechnology and Applied Biochemistry potential of this field has further been disclosed by the development of artificial metalloenzymes able to catalyze reactions with unknown natural counterparts [5,[20][21][22][23][24].…”