Many adverse environmental effects are associated with the industrial production of commodity chemicals like ammonia (NH3) from atmospheric dinitrogen (N2). Nitrogenase is the only enzyme capable of reducing N2 gas to a bioavailable form. Remarkably, the two-component protein system nitrogenase achieves this reaction at room temperature and atmospheric pressure, using ATP as the energy source. Though biological nitrogen fixation has been studied for nearly a century, the precise mechanism by which Nature activates N2 remains elusive. The overarching goal of this dissertation is to understand the molecular mechanism of biological nitrogen fixation, as this will lead to the development of more environmentally sustainable catalysts for industrial N2 activation and accelerate efforts to engineer the ability to fix nitrogen into plants.

Specifically, protein-protein interactions between and conformational changes within the nitrogenase component proteins are investigated in this work to gain insight as to why biological nitrogen fixation requires a specific, ATP-dependent electron donor protein to support catalysis. Through the study of site-directed nitrogenase mutants, a negatively-charged patch on the surface of the electron donor protein and a positively-charged patch on the surface of the catalytic component protein of nitrogenase were found to associate during turnover en route to the formation of a complex capable of interprotein electron transfer. While changes in the abilities of these mutant proteins to reduce H+ to H2 and C2H2 to C2H4 were explained well by the consensus Thorneley-Lowe model for nitrogenase reactivity, the model could neither predict nor explain the behavior of either wild-type or mutant proteins under an N2 atmosphere, performing the biologically relevant reduction of N2 to yield NH3 and H2.

Finally, the possibility for the electron donor protein in nitrogenase to transduce structural changes within the catalytic protein component upon binding is evaluated, and a mechanism for conformational gating of electron transfer in nitrogenase proposed. The findings of this dissertation would not have been achieved without significant efforts to create, optimize, and improve experimental methods in molecular biology and analytical biochemistry related to the study of nitrogenase, which are the subjects of the final chapters in this dissertation.

From only 20 amino acid building blocks, nature has designed and refined protein structures for a wide variety of specific purposes. Each protein molecule carries out a specific function in nature (structural component, transportation, catalysis, among myriad others) that is directly related to the structure of protein. Despite this impressive diversity, many proteins do not operate alone, but combine with other molecules—lipids, carbohydrates, nucleic acids, co-factors, and other proteins—to fulfill their functional roles. Consequently, understanding the structural properties of proteins and their interactions with other types of macromolecules provides insights for creating advanced protein-based hybrid components with tunable properties. Synthetic polymers are a large class of macromolecules that stand out in terms of their chemical diversity, tunable composition, controlled length (and thus physical properties) and easy acquisition. Thus, synthetic polymers have been commonly employed to augment the functional properties of proteins as well as creating hybrid materials with different proteins. Here, we have utilized synthetic polymers in a new context, namely in combination of protein crystals, to create a novel form of materials that seamlessly combine the advantages of proteins (functional diversity and atomically precise tailorability), crystalline materials (structural order and coherence) with those of synthetic polymers (flexibility, dynamics and stimuli-responsiveness). In this dissertation, we first report the combination of rigid/ordered ferritin crystals with superabsorbent polymers to create dynamic, self-healing, stimuli-responsive polymer-integrated-crystals (PIX). The so-formed hybrid materials possess the flexibility of the polymer components and the structural order of the protein crystals, and the macroscopic dynamicity arising from these two components can be controlled by changing the pH and ionic strength (Chapter 2). Following the initial success of the first PIX, we sought to expand the scope of utility for PIX materials by investigating the behavior of PIX by systematically varying polymer composition, ferritin surface charge, crystal packing, and identity of the crystallized protein. We carried out experiments and simulations to understand the individual effect of different components on the system. With a better understanding of the PIX system, we demonstrated that the properties of the PIX can be tuned by controlling pH and ionic strength, as mentioned above (Chapter 3). Finally, we showed that the original PIX system can be used as a platform for controlled encapsulation/release of target biomolecules in a size-selective fashion (Chapter 4). Overall, we have created a novel class of materials with unique physical and mechanical properties that possess tremendous potential for use in applications such as molecular storage/delivery and compartmentalized chemical reactions.

Proteins are the premier building blocks that Nature uses to create sophisticated systems. From carrying out the biological processes essential for life (e.g., photosynthesis, DNA replication, nitrogen fixation, etc.), to serving as mechanical supports for cells (e.g., cytoskeleton, membrane proteins, etc.), proteins play diverse, functional roles. Due to these capabilities, the synthesis of protein-based materials has become a burgeoning field that aims to introduce new-to-nature functionalities. Notably, researchers have recently begun to view protein crystals as viable candidates to serve as molecular templates. While historically used for structural analysis, protein crystals have more recently garnered attention as hosts for various extrinsic cargos including small molecules, nanoparticles, and other guest proteins. The Tezcan Lab, in particular, has successfully integrated synthetic polymers into crystals composed of Human Heavy-chain Ferritin (HuHF), resulting in a material termed Polymer-Integrated Protein Crystals (PIX). These hydrogel-crystals exhibit unprecedent characteristics such as reversible expansion/contraction and self-healing, all while maintaining crystallinity. Herein, we have focused on exploring the potential applications and limitations of PIX. In this dissertation, we elucidate how the different components of PIX can be modified to generate a multifunctional material. Starting with the building block, we created a mutant HuHF variant that exhibits a cysteine residue on the protein surface. By labeling HuHF with a specific small molecule, control over its crystallization behavior was achieved, leading to the formation of two distinct lattice arrangements. The spatial organization of HuHF molecules subsequently influences polymer distribution during PIX formation. Through these experiments, we discovered anisotropic polymer distribution can grant PIX with directional actuation during expansion/ contraction (Chapter 2). In another study, we examined how the dynamicity of PIX can be leveraged to encapsulate macromolecular cargo. By screening different proteins and encapsulation conditions, we revealed PIX can uptake guest proteins with high loading efficiencies (up to 46% w/w). Moreover, we explored the pH-dependency of cargo uptake and release using two model proteins, cytochrome c and lysozyme (Chapter 3). Lastly, we developed core-shell protein crystals using two distinct HuHF variants. This unique spatial organization of the HuHF molecules facilitated selective modification of the crystal surface. Due to PIX’s ability to encapsulate protein, we further functionalized the interstitial space and created a cell-like system (Chapter 4).

Metal ions are indispensable to biological function, as they are utilized for a myriad of processes encompassing signaling, electron transfer, and catalysis. These functions are made possible by exploiting intrinsic properties of the metal ions and the ligands to which they bind, represented by small molecules and metalloproteins. From the perspective of a synthetic chemist, proteins can be conceived as “macromolecular ligands”. Like those of small molecule ligands, the properties of “macromolecular ligands” can be parameterized at two discrete coordination spheres. The primary sphere is composed of the amino acid residues and exogenous ligands that coordinate the metal ion, and thus has the most direct impact on metal-based function. The secondary sphere is composed of residues that form mainly non-covalent interactions—hydrogen bonding, hydrophobic, and van der Waals (vDW) interactions—with the primary sphere. Although secondary sphere features do not directly bind the metal ion, they play an indispensable role in controlling metal-based reactivity. For example, the oxygen-binding affinity of myoglobin is influenced significantly by hydrogen bonding interactions between a histidine residue in its secondary sphere and the oxygen ligand that binds the heme co-factor. The large functional scope of metalloproteins is a testament to the wide diversity of primary and secondary spheres, and the efficiency with which metalloproteins execute these functions is a testament to the intricate interplay between the spheres. The functional potential of natural metalloproteins has long inspired protein design efforts. Two major metalloprotein design approaches are rational and de novo design. In rational design, primary and/or secondary sphere features of a natural protein are repurposed to mediate non-native functions. This approach has culminated in the engineering of new-to-nature functions spanning metal-hydride mediated ketone reduction and carbon-silicon bond coupling. While rational design represents an effective path to engineer metal-based functions, such functions are achieved within rigid and highly evolved protein folds/interfaces. Therefore, important questions remain unanswered: how does metal-based function emerge from an initially nonfunctional metalloprotein, and what are the minimum primary/secondary sphere coordination requirements to achieve metal-based functions? The work described in this dissertation stems from a de novo design approach in which both the quaternary structure and metal coordination site(s) of a metalloprotein are designed from scratch. The building block of our designed metalloprotein structures is cytochrome cb562, a natively monomeric, four-helix bundle protein. With cytochrome cb562 as our starting point, we obtain a diverse array of metalloprotein assemblies that serve as platforms for pursuing complex metal-based functions encompassing redox-based signaling and oxygen activation. In Chapter 2, we describe the sequential design of three metalloprotein trimers, TriCyt1, TriCyt2, and TriCyt3. TriCyt1, our initial trimeric construct, is obtained through a single hydrophobic mutation (G70W) at the interface of a cytochrome cb562 variant. Solution experiments indicated that TriCyt1 assembled as a trimer in the presence of all mid-to-late first row transition metals (MnII-ZnII), with yields ranging from 12% (+MnII) to 89% (+CoII). Crystal structures of NiII and CuII-supplemented TriCyt1 revealed a biologically rare His6 coordination motif. The only known natural His6 motif is present in calprotectin, a metal sequestering protein that coordinates MnII with nanomolar affinity. Motivated by the prospect of obtaining from scratch a MnII:His6 site of such high affinity, which had not yet been achieved in protein design, we sought to increase the preorganization of this metal coordination site through redesign of the C2 and C3 interfaces of TriCyt1. Computationally prescribed, mostly hydrophobic mutations at the C3 interface led to TriCyt2, a construct that trimerized with near-quantitative yield in the presence of MnII. A second round of redesign, in which salt bridges were installed at the C2 interface, led to a metal-independent, pH-switchable trimer which bound MnII with ~ 50 nM affinity. While a His6 primary sphere is suitable for achieving high affinity metal binding sites, our ability to design metalloproteins with broad functional potential hinges on our ability to engineer multiple types of metal binding sites. In Chapter 3, we describe our efforts to diversify the primary and/or secondary spheres of the TriCyt scaffold in pursuit of metalloprotein constructs which could coordinate lanthanides with high affinity and selectivity, mediate metal-dependent catalysis, and stabilize multinuclear metal coordination sites. The TriCyt series illustrates metal-ion-identity-independent assembly: the assembly path converges on the same architecture with the same metal coordination motif regardless of the metal ion added. In the TriCyt series there is low cooperativity between metal-ligand and non-covalent interactions, as metal coordination preferences play a minimal role in directing assembly. In Chapter 4, we describe our design and characterization of a variant of Rosetta interface design cytochrome 1 (A74/C96RIDC1) in which the cooperative interplay between metal-ligand and non-covalent interactions could be tuned by the redox state of the protein. In the oxidized state, C96-C96 disulfide bonds rigidify a tetrameric architecture (A74/C96RIDC14ox) whose assembly is independent of metal ion identity. Chemical reduction of the disulfide bonds gives rise to A74/C96RIDC1red, whose assembly is governed in larger part by the metal coordination preferences of exogenous metal ions. Whereas A74/C96RIDC14ox can only access two structural states (apo and metal-bound), A74/C96RIDC1red can access three distinct, metal-ion-identity dependent structural states with unique coordination environments. With five structural states whose accessibility is dependent on solution redox potential and metal ion identity, A74/C96RIDC1 represents a rare protein construct whose oligomerization path is varied through two different types of stimuli. We characterize the oligomeric and conformational states of A74/C96RIDC1 under disulfide-oxidized and disulfide-reduced states and in the presence of different mid-to-late first row transition metal ions, both in solution and via X-ray crystallography. We also employ density functional theory (DFT) and Rosetta interface calculations to attempt to quantify the energetic contributions of metal-ligand and non-covalent interactions and rationalize how differences in metal ion identity result in divergent metal-directed assembly paths.

Proteins are one of the main building blocks of life. Among their numerous functions are roles as biocatalysts for essential chemical reactions, signaling agents that coordinate communication down to the sub-cellular level, and as modules for constructing cellular superstructures. In many cases, these functions rely on the ability of proteins to form stable and specific associative interactions with other proteins, as well as with metal ions. Drawing inspiration from nature, we deploy these functional features as design elements that aid in the construction of artificial metalloprotein assemblies. Using an iterative design approach, we have engineered the monomeric protein cytochrome cb562 by application orthogonal strategies: Metal Designed Protein Self-Assembly (MDPSA), Metal-Templated Interface Redesign (MeTIR), and the installation of intermolecular disulfide bond crosslinks. This synergistic design approach afforded a self-assembling protein variant, C38/C81/C96R1, which bears metal chelating groups, a designed dimerization interface (termed i1), and surface-exposed cysteine residues. In the presence of ZnII this engineered variant self-assembled into a tetramer, Zn-C38/C81/C96R14. Removal of ZnII from the assembled tetramer resulted in hydrolysis of a single C38-C38 disulfide bond, leaving the five remaining crosslinks intact. Thus, C38/C81/C96R14 is an allosteric protein, capable of remotely coupling ZnII binding at is central binding sites to the breakage of a peripheral disulfide bond. In this work, we discuss the design of the C38/C81/C96R14 protein as well as the demonstration of this allosteric behavior. We further carry out biochemical and biophysical characterization of this protein and related variants to determine the structural and energetic basis of this Zn-disulfide allostery. The C38-C38 disulfide bonds are embedded in the i1interface which, crucially, forms malleable protein-protein contacts. The flanking disulfide crosslinks of C38/C81/C96R14 serve as structural conduits for coupling the pair of i1 interfaces, which is critical to develop the requisite driving force to effect disulfide bond hydrolysis. In this designed system, it is the underlying malleability of the structure that gives rise to the allosteric behavior. We consider this as a successful application of synergistic design to give a coordinated protein function, and anticipate that adoption of similar design paradigms will greatly benefit ongoing protein engineering efforts in the community at large.

Proteins are nature’s chosen building blocks to create sophisticated and functional 0-, 1-, 2- and 3D assemblies that are necessary for the complexity of life. Accordingly, there is significant interest in the development of highly ordered supramolecular protein arrays that mimic or even surpass the complexity and functional versatility of those assemblies found in nature. However, due to the chemical and structural heterogeneity of proteins, the rational design of supramolecular protein architectures is challenging. In contrast, coordination polymers such as metal-organic frameworks (MOFs) benefit from a high degree of modularity, where individual components can be interchanged to produce structurally unique architectures with vastly different materials properties. Inspired by the synthetic versatility of MOFs, we developed a method for assembling protein oligomers into highly ordered frameworks, termed protein-MOFs. Akin to more conventional MOFs, the modularity of these lattices stems from the multiple interchangeable components. We engineered a protein node through the installation of outward-facing metal-binding sites at the C3 vertices of the symmetric cage-like protein, human heavy-chain ferritin. These sites coordinate a variety of transition metals with high fidelity, allowing for the formation of extended crystalline networks when connected by ditopic bridging ligands. The symmetry of the resulting crystalline lattice is dictated by the inherent symmetry of the protein, the coordination preferences of the metal, and the geometry of the bridging ligand. This modularity led to the discovery of stimuli-responsive protein-MOFs capable of dynamic crystal-to-crystal transformations. Protein-MOFs have the potential to be utilized for the construction of functional biomaterials.

Proteins are Nature’s fundamental multitools, fulfilling crucial roles in catalyzing complex chemical reactions, mediating cell-cell signaling to coordinate biochemical responses, and providing the structural scaffolding necessary for intracellular transport and cell motility among a myriad of other functions. The functional diversity of proteins is enhanced by the associations of intracellular protein, nucleic acid and small molecule components to generate sophisticated self-assembled architectures. The “bottom-up” construction of biological components is a burgeoning field of study which seeks to generate novel functional assemblies by directing protein interactions in a controlled fashion. Protein complexes in Nature are driven by an accumulation of weak noncovalent interactions over large interfaces, which ensure specific and stable assembly of the desired architecture. However, such nuanced interactions are difficult to emulate by intuition (or computation), making their designability one of the foremost challenges in protein engineering. Nevertheless, our strategies streamline such design efforts via the integration of well-studied biological motifs into self-assembling protein scaffolds to generate structurally and functionally diverse architectures.

Previous studies have shown that reversible yet specific interactions, such as metal-coordination and disulfide bonding, can be used to programmably assemble both discrete and pseudoinfinite protein oligomers. We first utilized designed crystalline lattices to generate functional materials through post-translational modification of assembled proteins using biological enzymes. Incorporation of a functional peptide substrate onto our protein scaffolds enables the use of phosphopantetheinyl transferase (PPTase) enzymes to site-specifically tailor the surface of crystalline two-dimensional protein materials. In addition to expanding the functionality of existing designed assemblies, we explore the use of biologically relevant motifs to create novel protein-based architectures. Integrated protein and nucleic acid (NA) complexes are among the most complex biological machines in Nature, but the design of a synthetic assembly of protein and NA components via synergistic interactions remains an outstanding challenge in biomolecular design. We create a protein-DNA conjugate via covalent tethering of a monomeric protein and single-stranded DNA to enable the assembly of an artificial three-dimensional nucleoprotein architecture through protein-metal coordination, Watson-Crick DNA base pairing, and DNA-protein interactions. Appropriately balanced thermodynamics of these interactions is necessary to achieve well-ordered self-assembly products instead of disordered protein-NA aggregation (as we observe when one set of interactions dominates). Finally, we use siderophore-inspired hydroxamate motifs, which selectively bind Fe3+ ions very tightly, to construct bimetallic protein cages from a monomeric protein building block. We show that a protein monomer modified with both hydroxamate groups and zinc-binding motifs assembles through concurrent binding of Zn2+ and Fe3+ ions to form dodecameric and hexameric protein cages. These cages can assemble and disassemble in response to multiple stimuli, and can be used for cargo encapsulation and storage. Overall, we show that the integration of native biological components and protein design strategies enables the construction of novel functional protein assemblies that can serve to guide future protein engineering efforts.

Nitrogen is an essential component of many biomolecules such as DNA and proteins. Biological nitrogen fixation is carried out by the enzyme nitrogenase. This multi-electron reduction requires the precise timing of electron and proton flow from the reductase, Fe-protein, through the [8Fe-7S] P-cluster to the catalytic site, FeMo-cofactor, located in the catalytic protein, MoFe-protein. Electron flow through nitrogenase is a dynamic process controlled by a conformational “gate” in which structural changes in MoFe-protein promote the accumulation of electrons at the FeMo-cofactor. Elucidating the mechanism thus requires a detailed understanding of structural changes that occur during catalysis. I have investigated the dynamics involved in nitrogenase catalysis, focusing on (1) the P-cluster’s dynamic role in orchestrating electron transfer to FeMo-cofactor and (2) structural characterization of the nitrogenase complex during N2 reduction. The experimental results discussed in Chapters 2 and 3 of this dissertation describe how the P-cluster has evolved to rest on the brink of stability, and that mutations to the redox-switchable, O-based P-cluster ligand (serine or tyrosine) result in a cluster that can reversibly lose Fe atoms. Furthermore, the O-based ligand protects the P-cluster from oxidative stress in vivo, is required for efficient diazotrophic growth under Fe-limiting conditions, and protects the P-cluster from metal exchange in vitro. These results indicate the native flexibility of the P-cluster are vital to its function in timing electron transfer to the active site. In Chapter 4, I discuss structural characterization of the nitrogenase complex at high-resolution during catalytic N2 reduction with cryoEM. Many structures were determined: structures of the complex during turnover at 2.6Å and 2.7Å, free MoFeP at 1.9Å, and ADP.AlF4—-inhibited complexes at 2.4Å and 2.8Å. Taken together, these structures have provided valuable insights into the mechanism of biological nitrogen fixation. The complex formed during turnover has a 1:1 Fe-protein:MoFe-protein stoichiometry, implying that there is negative cooperativity between the two halves of the complex. Furthermore, conformational differences between the two halves of MoFe-protein reveal previously unobserved conformational changes, which may be a part of the conformational “gate”. These observations provide critical insights into the dynamics required for nitrogenase function.

For over 30 years, porous silicon as a material has been leveraged for its usefulness in biomedical and sensing applications. Its tunable structural features, low toxicity profile and readily modifiable surface render this material extremely useful for a wide variety of applications. By chemically modifying surface species, such as silicon hydrides and silicon oxides with silanes, the properties of porous silicon can be enhanced for its use for chemical sensing, biomedical imaging, and drug delivery.

After a brief introduction to porous silicon materials, the first part of this dissertation details surface-modified porous silicon photonic crystals for the chemical sensing of toxic vapors and nerve agents. Chapter 2 utilizes a dual-peak porous silicon photonic crystal embedded with specific for the selective detection of hydrogen fluoride (HF), hydrogen cyanide (HCN), and the chemical nerve agent diisopropyl fluorophosphate (DFP). The pore walls are rendered hydrophobic with octadecylsilane to aid with the loading of the colorimetric molecules while being insensitive to humidity fluctuations. This provides a robust means to develop a remote detection system for chemical agents. Chapter 3 employs the same photonic crystal, modified, however, with a specialized protein-based gatekeeper that is rendered semi-permeable only in the presence of HCN. This is one of the first novel designs of a bio-inorganic sensor capable of detecting chemical agents with high specificity and precision.

The second portion of the dissertation describes how surface-modified porous silicon nanoparticles can be applied in biomedical applications. The first project details the use of Anti-KIT protein DNA-aptamers decorated onto a fluorescently labelled porous silicon nanoparticle for the in vitro and in vivo imaging of gastrointestinal stromal tumors. This work provides an effective platform in which aptamer-conjugated porous silicon nanoparticle constructs can be used for the targeted imaging of KIT-expressing cancers. The final project utilizes hydrophobic porous silicon nanoparticles for the delivery of erucamide, a highly hydrophobic fatty acid amide, within the retina. By harnessing the versatility of porous silicon, erucamide’s target cells and mechanism of neurotrophic action can be identified.