Proteins are essential macromolecules for all living organisms. They provide cellular

structure and perform most of the metabolic functions essential for all life. The importance of

proteins makes them the most studied and exploited macromolecules. We can exploit the

structure of a protein to design a specific therapeutic to treat a disease or we can use proteins as biocatalysts for the efficient creation of molecules. In many instances, these applications of proteins are difficult to achieve. The work in this dissertation focuses on the development and evaluations of novel techniques to aid in the study and use of proteins.

The first part of this dissertation focus on the creation of a series of symmetric oligomers

to be used as crystallization scaffolds. Such scaffolds are intended to induce their symmetry onto asymmetric protein crystallization target proteins. The ability to determine the crystal structure can be essential for the creation of new targeted drugs or the better understanding of a biological process. Unfortunately many proteins fail to crystallize for reasons that are not well understood. It is thought that such induction of symmetry and variety of geometrically distinct scaffolds will aid in the crystallization of difficult-to-crystallize proteins. Preliminary results of these novel scaffolds and existing scaffolds are described.

In the second part, applications of symmetric scaffolds for the creation of enzymatic materials are presented. These purely proteinaceous assemblies are designed to replicate previous

described enzyme encapsulating materials. These materials typically improve enzyme reaction rates and product extraction. The final part of the dissertation focuses on the shell protein PduA from the 1,2-propanediol-utilization bacterial microcompartment (MCPs). These MCPs encapsulate metabolic pathways and contain volatile or toxic pathway intermediates. Research into turning these MCPs into bioreactors containing non-native enzymes is ongoing in many labs. Full realization of this technology relies on the encapsulation of new metabolic enzymes and transport of novel substrate and products through the shells. These processes are poorly understood, here structural studies of shell protein permutations. These permutations alter the topology of the shell protein allowing the scaffolding of proteins to the exterior surface of the MCPs. Finally, the efforts to elicited the interaction of specific targeting sequences to shell protein by x-ray crystallography are discussed.

Protein nanoparticles are self-assembling, symmetric, supramolecular protein structures with diverse applications that have significantly furthered the fields of structural biology and molecular biology. Within structural biology, both the size limitation of cryogenic electron microscopy (cryo-EM) and preferred specimen orientation have proven to be outstanding challenges in understanding the protein structure of small protein targets. Similarly, there is a deficit of robust methods for imaging proteins and protein complexes at high resolution within the cellular context. Here, I describe the development of protein nanoparticles, or protein cages, as rigid, symmetric scaffolds capable of resolving the structure of small proteins with cryo-EM and as fluorescent, fiducial biomarkers for targeted cellular imaging with confocal microscopy. This study underscores the profound impact of protein nanoparticle technology and its capacity to further revolutionize biochemical research.

Over the early 21st century, structural biology has laid a robust foundation for our understanding of protein molecules. The atomic principles of their structure, how their sequence specifies such a structure, and conversely, how to enumerate an amino acid sequence to adopt a fold, are problems each closer to solved than not. Once the domain of material science and chemistry, materials of various shapes and sizes can now be made out of protein precursors. Designed protein building blocks have been synthesized that self-assemble into tetrahedral, octahedral, and icosahedral molecular cages, as well as infinitely ordered lattices such as two dimensional layers and three dimensional crystals. This thesis provides descriptions used to both create and apply protein nanomaterials towards the study of biochemical phenomenon. Particular interest is given to methods of searching for native like contacts and emulating their assembly into defined quaternary structures. By taking inspiration from nature and utilizing hypothesis driven symmetric materials engineering, I demonstrate the creation of proteins which form new materials in the laboratory and methods to engineer existing materials, including a high resolution imaging scaffold. These advances remove barriers to nanomaterials development and deepen the understanding of protein molecules.

ABSTRACT OF THE DISSERTATIONTheory, Design and Characterization of Protein Symmetry Combination Materials by Joshua Laniado Doctor of Philosophy in the Molecular Biology Interdepartmental Doctoral Program: Biochemistry, Biophysics & Structural Biology University of California, Los Angeles, 2020 Professor Todd O. Yeates, Chair

Nature has evolved a plethora of sophisticated protein complexes to carry out fundamental biological processes. While most of these exquisite macromolecular machines exhibit complex architectures, many are composed of only a few different types of subunits. Understanding how protein molecules combine to form these remarkable self-assembling structures only makes sense in the light of symmetry. By limiting the number of distinct interactions required between individual subunits, symmetry offers a simpler route for the evolution of supramolecular assemblies such as viral capsids and bacterial microcompartments. Principles of symmetry and self-assembly have invigorated recent efforts in molecular engineering giving rise to a growing suite of novel protein materials such as finite cages and extended crystalline arrays. These designed assemblies are rapidly finding applications in areas as diverse as vaccine design, atomic imaging, enzyme scaffolding and molecular delivery. Despite significant advances in computational approaches and design strategies, constructing these materials remains extremely challenging. Here, we address key experimental and theoretical limitations to improve the prospects for the routine design of novel symmetric protein materials. In Chapter 1, we review current methodologies for designing self-assembling protein nanomaterials. A first approach presented the idea that when two separate symmetric oligomers associate in some geometrically defined way, a structure with higher symmetry can be obtained through self-assembly. There, an alpha-helical linker is used to connect two oligomeric components and to control their relative geometry. A second approach does not involve genetic fusion but relies instead on the computational design of a novel protein-protein interface. After reviewing the successful constructions resulting from both methods, challenges and limitations are discussed. In the fusion approach, the inherent flexibility of the alpha helical linker can lead to the formation of unintended assemblies. Alternatively, the interface design strategy exhibits limited success in predicting viable protein interfaces. The prevalence of such limitations dramatically hinders the creation of novel materials, motivating the development of alternate strategies. In the next chapter, we introduce a new approach for the design of symmetric self-assembling nanomaterials. Building upon the fusion approach, the original alpha-helical linker is replaced with a heterodimeric coiled coil as an attempt to reduce flexibility. Further, the use of a known heterodimeric interface to combine component oligomers alleviates the challenges associated with de novo interface design. Ten symmetric protein cages were designed using this method among which two were structurally characterized. One design assembled as intended while the other crystallized in an alternate form. Geometric distinctions between the two help explain the different degrees of success, leading to crucial lessons and establishing clearer principles for the creation of novel nanoscale protein architectures. While some experimental aspects have been addressed, only a small fraction of the possible design space has been explored. That space, which is anticipated to offer a multitude of symmetry-based combinations, has not been described in theory. In Chapter 3, we articulate all of the possible kinds of protein-based materials that can be created by combining two symmetric oligomers. Specifically, 13 types of cages, 35 types of 2-D layers and 76 types of 3-D crystals are identified as possible targets for design. We lay out a complete rule set for constructing all such symmetry combination materials (SCMs) and introduce a unified system for parameterizing and searching the construction space for each case. This theoretical and computational study provides a blueprint for a blossoming area of macromolecular design. Owing to the complexity and our limited understanding of the rules that govern protein behavior, designing protein-protein interfaces remains challenging. Current approaches rely on empirical or knowledge-based energy functions and optimization algorithms that often fail to produce stable interfaces. On the other hand, there is growing evidence that the database of known protein structures is now sufficiently large to cover the structural landscape of protein interfaces. In Chapter 4, we argue that carefully-selected structural motifs can be used as templates for interface design. We introduce Nanohedra, a fragment-based docking tool that harnesses the power of our theoretical framework to enable the design of all possible SCMs. Prospective designs of symmetric materials are proposed along with a retrospective analysis of recent design studies. In this analysis, our tool recapitulates all successful designs while poorly ranking failed ones. With a user-friendly interface and a unified protocol for symmetric protein design, Nanohedra enables the creation of a universe of novel nanomaterials and opens new avenues for nanobiotechnology.

In nature, it is extremely common to find proteins that assemble into homo-oligomeric complexes from multiple copies of themselves. Almost half of known proteins form such complexes, most of which are cyclically or dihedrally symmetric. In some exceptional cases, however, protein molecules will self-assemble into much larger, closed three-dimensional geometries resembling the Platonic solids. Examples include icosahedral viral capsids, bacterial microcompartment shells, and octahedral ferritin assemblies. Protein scientists have studied and marveled at these exquisite protein cage structures for decades, and some have even ventured to produce novel types of protein cage assemblies unseen in nature through their own engineering efforts. In recent years, the field of protein design has seen striking progress in the development of design methodologies for taking proteins found in nature and modifying them to self-assemble into cages of tetrahedral, octahedral, or icosahedral point group symmetry, and these unique new types of protein assemblies are even beginning to find use in medicine, imaging, and biomaterials applications. My thesis work addresses both the design and application areas of the field of symmetric protein cage design.

In Chapter 1, I include a recent review article on high symmetry protein assemblies, both natural and designed. A survey of all known structures in the Protein Data Bank that self-assemble into unique complexes with tetrahedral, octahedral, or icosahedral symmetry gives context for the types of biological functionality that seem to necessitate or benefit from such higher-order symmetries, although some intriguing mysteries remain unsolved. Our comparison of natural protein assemblies to the recent types of designed protein cages also emphasizes some unique properties of designed cages that remain unseen in natural assemblies.

Next, I go on to describe some recent efforts to improve cage design methods to make cages that more reliably self-assemble into desired architectures when produced in the laboratory. In Chapter 2, we describe the design and characterization of two tetrahedral protein cage assemblies which were engineered to have hydrogen bonding networks at the interface between their two oligomeric components. These cages exhibit exceptionally high levels of soluble expression compared to most previous designed cages, but atomic structures solved by X-ray crystallography reveal some surprising deviations from the designed models.

In Chapter 3, I describe efforts to design and characterize a protein icosahedron that self-assembles from 60 copies of a single designed protein subunit. To date, a designed icosahedral protein assembly formed from genetically fused protein oligomers (as opposed to multiple proteins self-assembling with a computationally designed interface) has yet to be validated in atomic detail. Challenges in achieving this goal have made it clear that novel, alternative design strategies are necessary. We describe the creation of a double-fusion protein containing dimer-, trimer-, and pentamer-forming protein domains in a single protein construct, which forms an icosahedral assembly when overexpressed in bacteria. The cage assembly is characterized by electron microscopy, small angle X-ray scattering, and other solution-state methods.

I then go on to describe a project which applies a previously characterized tetrahedral protein cage scaffold as a platform for the multivalent display of cellulase enzymes. In Chapter 4, we describe the utilization of the T33-21 cage scaffold as a platform for covalently fusing other proteins to the exterior of the cage post-translationally using a sortase ligation method. In this work, we attach two different cellulase enzymes simultaneously to the cage scaffold and demonstrate increased synergy between the two enzymes in cellulose degradation assays.

In conclusion, the work described in this thesis contributes to the ongoing development of novel design methodologies for engineering high symmetry protein cages, including some important lessons learned along the way, and goes on to describe the application of a designed cage scaffold as a multi-enzyme display platform for potential use in biofuels and other biomaterials technologies.

Bacterial microcompartments, or MCPs, are supramolecular structures analogous to eukaryotic organelles. These assemblies are understood to organize and compartmentalize the cytosol by sequestering and optimizing a myriad of metabolic reactions. They enable the diffusive transport of substrates, acting as a semi-permeable barrier that prevents the efflux of toxic intermediates. Unlike their membrane-bound eukaryotic counterparts, MCPs are comprised entirely of a proteinaceous outer shell. The so-called BMC domain (for bacterial microcompartment) is the canonical building block for all MCPs. BMCs oligomerize to form hexamers disks which tesselate laterally to form the nearly flat facets of the outer MCP shell. Pentameric proteins form the polyhedral vertices. Despite persistent research in the field, bacterial microcompartments remain poorly understood. This work seeks to enhance our foundational knowledge of MCPs through structurally characterizing BMS shell proteins and highlighting their potentially specialized functions, presenting a novel structural resource that organizes existing structural data and recent work that begins to reveal the native topology and long-range organization of MCP shells.

The chapters of this thesis describe the structure and function of three proteins involved in assembly and function of bacterial microcompartments. Bacterial microcompartments (MCPs) are often thought of as bacterial organelles; they are proteinaceous structures that spatially compartmentalize metabolic reactions for increased catalytic efficiency. There are three types of characterized MCPs: those that catalyze either the catabolic utilization of propanediol (Pdu MCP), ethanolamine (Eut MCP), or carbon dioxide (called carboxysomes).

Chapter 1 describes studies of bacterial microcompartment vertex proteins (BMVs) from various microcompartments. BMVs are thought to occupy the 12 vertices of the MCP icosahedron capsule. However, past structural studies of the BMV from E. coli, EutN, reveal pseudo–hexagonal shapes, whose geometry is unfit for incorporation into pentagonal vertex spaces of microcompartments. We developed a novel method called OCAC (Oligomeric Characterization by Addition of Charge) to probe BMV oligomeric states in solution. We also endeavored to determine the oligomeric states of the two conserved BMV proteins from alpha–carboxysome operons. The reason for the existence of paralogous BMV proteins in alpha–carboxysome is not understood. The contents and figures of Part–I of Chapter–I were published in Protein Science in 2013 (1). Yuxi Lui performed the experiment reported in Figure 2. Soheil Gidaniyan and Nicole Wheatley performed protein expression, purification and crystallization of GrpN and other required proteins. Duilio Cascio provided assistance in solving the GrpN crystal structure. Nicole Wheatley and Todd Yeates coordinated and designed experiments and prepared the manuscript. The contents of Part–II have not been published. Joanna Ngo expressed, purified and performed gels shown in that chapter sub–section. Nicole Wheatley designed and coordinated efforts. Chapter 2 describes the discovery of a novel Rubisco (Ribulose–1,5–bisphosphate carboxylase/oxygenase) chaperone encoded within the operons of alpha–carboxysomes. We named this distant pterin–4a–carbinolamine dehydratase homolog acRAF, for alpha carboxysome Rubisco Assembly Factor. Heterologous co–overexpression of acRAF, GroELS and CbbLS yields greatly increased amounts of soluble, folded Rubisco compared to co–overexpression with GroELS and CbbLS alone. The contents and figures of Chapter–II were published in the Journal of Biological Chemistry in 2014 (2). Christopher Sundberg created the DNA constructs used in Figure 5 of that publication, or Figure 9 of this thesis. Soheil Gidanyian and Nicole Wheatley performed protein expression, purification and crystallization experiments on acRAF. Nicole Wheatley performed the experiment shown in Figure 10A and AB of this thesis. Soheil Gidanyian performed experiment shown in Figure 10C. Duilio Cascio assisted in solving the crystal structure of acRAF. Todd Yeates made Figure 7A. Nicole Wheatley and Todd Yeates prepared the manuscript. Nicole Wheatley designed and coordinated the efforts of the work. Chapter 3 describes a nitrogen regulatory PII–like protein encoded within several proteobacterial alpha–carboxysome. Crystal structures of this protein, here named ndhPII, display several unique characteristics compared to canonical nitrogen regulatory PII proteins. We structurally analyze both unbound and ADP–bound ndhPII crystal structures, and speculate about its regulation of inorganic carbon assimilation in these organisms. The work contained in this chapter is not yet published. Joanna Ngo and Nicole Wheatley performed protein expression, purification and crystallization of ndhPII. Duilio Cascio and Michael Sawaya solved and refined both crystal structures. Nicole Wheatley designed and coordinated the efforts of this work.

All aspects of life are controlled by proteins which often assemble into complex arrangements. Research on natural and unnatural protein assemblies enhances our understanding of biological science. The research described here provides structural and mechanistic insights into how protein assemblies could be utilized to improve or develop novel protein functions. Bacterial protein assemblies, microtubule-targeting compounds, and designed protein assemblies were investigated with X-ray crystallography and our results may further the understanding of these targets in pursuit of therapeutics for cancer, obesity, and other conditions.

The first study revealed an unexpectedly large structure and putative mechanism for the enzyme DmrB that may help in the design of anti-obesity drugs targeting gut bacteria. A second effort helped to explain how the outer shells of huge bacterial microcompartments may assemble together. The research carried out may help better implement efforts underway to engineer more stable microcompartments for new biotechnology approaches. Structural studies of the interactions which mediate enzyme encapsulation in bacterial microcompartments were also pursued. Our results establish methods for the isolation and subsequent characterization of binary and potentially ternary complexes of microcompartment shell proteins and luminal enzymes.

Current tubulin-targeting cancer therapeutics suffer from many drawbacks underscoring the need to further understand the structure of eukaryotic protein assemblies such as microtubules. We describe the structural characterization of two recently-identified compounds that potently kill cancer cell lines. High-throughput screening identified MI-181 and C2 in contexts of mitotic inhibition and cancer cell-death. X-ray crystal structures of MI-181 and C2 bound to a complex containing αβ-tubulin, tubulin tyrosine ligase, and the tubulin-binding protein stathmin revealed details of how these compounds interact with β-tubulin subunits. These structures clarify the unknown binding properties and may guide the improvement of these compounds as candidates for therapeutics or chemical probes for better understanding cell division.

In addition to natural protein assemblies, unnatural symmetric protein materials can be achieved through protein design. We have solved crystal structures of four computationally-designed protein assemblies. Additional structural studies of designed proteins revealed structures of sequences engineered to form cyclic oligomers and three-dimensional lattice symmetries. These structures emphasize the design accuracy that can be achieved using computational methods in favorable cases. These most recent designed assemblies are built from two distinct components, a strategy that lends itself to versatile construction of functional nanomaterials for a variety of applications.

Furthermore, we have developed an additional new method to design two-component protein assemblies which may be engineered for new functions. Fusion of a self-associating heterologous pair of helical elements to symmetric oligomers can be implemented in a way that generates three-dimensional closed systems or extended arrays of protein. Protein engineering applications are proposed and intial designs are described for several biotechnology purposes.

Self-assembling protein designs will contain short peptide ligands for cancer-related growth receptors. Designs in which symmetric oligomers self-assemble into extended rod-like filaments were tested for this application. Proper assembly was investigated with electron microscopy after purification from bacteria. The coiled-coil fusion design approach identified candidates for novel nanomaterials which may induce cellular responses related to cell growth and cancer in cultured endothelial cells.

Finally, improving the properties of key enzymes can overcome limitations of in vitro metabolic engineering systems. Enzymes for synthesizing isoprene – a starting compound for generating a variety of value-added products – are a useful test-bed. We incorporated a dimeric plant enzyme for isoprene synthesis into the design of a single-layer array of self-assembling symmetric oligomeric enzymes. Two designs are described which enable the isolation of two components separately which may self-assemble upon mixing to form a two-dimensional layer.

Biological containers known as protein cages are widespread in nature—displaying unique functionality via their ability to compartmentalize molecules in an internal lumen volume. Composed of repeated copies of a small number of protein subunits arranged symmetrically, protein cages are relatively simple in composition, and yet complex in structure. In nature these assemblies function in transport and protection, storage of important molecular cargo, and sequestration of metabolic reactions. These demonstrations of incredible functionality have inspired researchers seeking to engineer complex protein architectures with diverse functionality to direct their attention towards symmetric protein cages. Resulting from these efforts, a modest number of protein engineering studies have succeeded in replicating these structures de novo in hopes of applying them to the fields of medicine and biotechnology; however, developing applications for these assemblies that outperform existing technologies remains a challenging exercise. The primary aims of my thesis work are the design and development of applications for symmetric protein cages with a particular focus on mimicking natural functionalities not yet recreated via artificial protein design methods. We note the limitations of prevalent protein design methods, and technological advances that may revolutionize the field.The first chapter of this thesis is a brief introduction to protein cages as platforms for engineering, as well as efforts to design them de novo in the laboratory. In the second chapter of this thesis we sought to advance on existing methods for protein cage engineering by designing new cages with novel interface construction. These cages incorporate a grafted interface onto each of two components making up the cages. In this work we describe challenges associated with direct rigid linkage of protein components via alpha helical extension and discuss whether the technique is a viable strategy for future cage design efforts. Next we attempt a novel approach to addressing a long-standing challenge in the field of protein design: engineering structurally responsive protein cages. The natural world holds numerous examples of protein cages that can assemble and disassemble in response to specific stimuli, but recreating this effect in the laboratory has proven challenging. In chapter three we present work on the design of protein cages that disassemble in response to a specifically chosen target protease. This work related to protein cage disassembly has implications in therapeutic design, as proteases are common therapeutic targets, and developing systems responsive to diseases biomarkers could be of great utility. In the next chapter we expand our focus on cage disassembly to other types of triggers for cage disassembly. In this work, we devise a system utilizing antibody-mimetic molecules called DARPins that allows for the design of protein cages that disassemble in response to any protein of interest. The work in this chapter focuses on a natural cage assembly (sulfur oxygenase) as a platform for development of this effect. The fifth chapter of this thesis covers work related to designing protein-cage based nanoparticles to be used as immunosorbents for patients on dialysis. The work addresses a clinical need for methods to remove the protein beta-2 microglobulin from the blood, as high concentrations are common in patients on long-term hemodialysis therapy, but can be toxic. We describe a protein cage that displays nanobodies on its exterior surface to generate beta-2 microglobulin adsorbent cages (BACs). In conclusion, the research performed in this dissertation work seeks to advance on existing methods for protein cage engineering by designing new cages with novel interface construction. I then used previously described artificial protein cages to demonstrate triggerable protein cages with dynamic assembly properties that are responsive to molecular stimuli, as well as developing of an immunosorbent nanoparticle with clinical applications.