Thesis (Ph.D.)--University of Washington, 2020Cyclic two-fold (C2) symmetric ligands are common in nature, synthetic chemistry, and medicine. Additionally, we can now design millions of C2 symmetric peptides with an incredible diversity of sizes, shapes, and chemistries. De novo proteins capable of binding these C2 symmetric ligands could be useful in various applications, but scaffolds and methods to do this have been lacking. To solve this problem, I created a diverse set of C2 symmetric proteins with central cavities. I first designed curved repeat protein monomers sampling a continuum of curvatures, and then docked these into homodimers, generating a very wide range of C2 cavity shapes and sizes for functionalization. In total, 77 scaffolds were experimentally characterized, and of these, 23 (30%) appear to be folded as designed based on Small Angle X-ray Scattering data. Furthermore, crystallographic data for 4 designs (2 scaffolds and 2 functionalized binders) confirms the proteins fold as expected. A third scaffold design was determined to be monomeric by crystallographic analysis. Despite its failure to form the designed homodimer, the solved monomer was in close agreement with the design model. We believe that these diverse scaffolds provide a rich set of starting points for binding a very wide range of C2 compounds. Advantages of this conception are that the cavities can be very diverse in size, shape, and available sidechain chemistry, and as the protein hydrophobic core is separated from the pocket because the cavity lining residues are on the exterior of the monomers, functionalization to create binding interactions for specific compounds is unlikely to destabilize either the monomers or the dimer interface. Finally, we used these scaffolds to bind symmetric chlorophyll dimers, which could have applications in synthetic light-harvesting, as well as to bind a C2 symmetric peptide, which could become a platform for the creation of entirely bioorthogonal chemically induced dimers
