UC San Diego

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.