UCLA

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.