We frequently find proteins exist in oligomeric forms in nature. The abundance of dimers, trimers and tetramers with cyclic or dihedral symmetries in the Protein Data Bank is a good testimony. Even more, it is not rare to find proteins form highly ordered, symmetric, large complexes. These oligomeric forms are usually essential for their functions. Ferritin forms an octahedral cage with 24 subunits to store iron; some virus capsid proteins assemble into icosahedral cages; vaults, which are large dihedral particles widely conserved in eukaryotes, have biological functions yet to be discovered. These fascinating structures inspire three types of questions: How do individual subunits interact form such symmetric complexes? How can we reproduce such complexes with protein engineering? How do we put engineered symmetric protein complexes to application? My thesis work consists of projects addressing all three questions.
My first project, described in Chapter 1, concerns bacterial microcompartments (MCP), which are large proteinaceous organelles enclosed by an icosahedral or pseudo-icosahedral shell. MCPs usually enclose special metabolic pathways that are inefficient or toxic in the cytosol. To do so, MCPs must form a sealed barrier with its shell proteins. It was hypothesized that at least one type of the proteins forming the shell of MCPs has to be pentameric instead of hexameric. Indeed, we proved that the BMV proteins, a family of protein highly conserved in MCP operons, formed pentamers in solution. Together with other crystallographic evidence, we conclude BMV proteins form pentamers to cap and seal the MCP shell. In addition to MCPs, I worked on another natural oligomeric protein, bactofilin. Bactofilins are fiber-forming proteins that are widely conserved among bacteria. These proteins have roles in diverse biological functions including but not limited to cell motility, cell wall synthesis and modification. Chapter 2 describe my preliminary biochemical and structural work on bactofilins.
Next, I moved on to symmetry-based engineering protein complexes. In Chapter 3, I included a recent review paper on the theory and successes in symmetry-based protein engineering that I participated in preparing. Designed complexes need to be validated at high resolution with X-ray crystallography, but for a long time, the low yield and solubility of the designs complicated their validation. In Chapter 4, we show that mutating solvent-exposed side chains to charged amino acids improved the solubility of a previously low yield tetrahedral design and enabled validation by crystallography. Next, I advanced to a bigger challenge in designing symmetric nanoparticles—icosahedral particles. Icosahedral particles are made up of 60 asymmetric units, as compared to 12 in tetrahedral particles, making them much more difficult to design with accuracy. I was able to validate three different icosahedral design with crystallography, making them the largest designed protein assemblies ever crystallized to date. This work is described in Chapter 5. Additionally, I have made other independent design efforts, one to combine DNA and protein as building materials to design tetrahedral complexes, another to design protein sheets with layer group symmetry. These efforts are documented in Chapter 6.I
In the last chapter, I utilized the validated tetrahedral designs as a scaffold in cryo-electron microscope (cryo-EM) for small targets. Despite recent advancements in cryo-EM techniques, small targets remain difficult. By arranging small targets around tetrahedral particles, we can overcome the size limit and provide multiple views to alleviate the commonly seen orientation preference. My project used a type of versatile adaptor protein, designed ankyrin repeat proteins (DARPins), to connect the tetrahedral particles to the imaging targets. We show that the resulting construct is amenable to structural analysis by single particle cryo-EM, allowing us to identify and solve the structure of the attached DARPin at near-atomic detail. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other proteins, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.
In conclusion, my thesis work contributes to the understanding of natural oligomeric complexes, expands our capacity in designing symmetric assemblies, and puts forward an example of a useful application of the designed assemblies.
