UC San Diego

The field of protein design strives to engineer new molecules that interact in a specific, controlled manner to form novel functional complexes. Engineered proteins that generate specific complexes upon the addition of an exogenous agent, such as metal ions, will likely be integral elements of these efforts. Molecular control over protein assembly and disassembly is possible through the introduction of novel metal-binding sites at precise locations in protein complexes. These methods have primarily generated metal-mediated and metal-controlled homodimers. The overall goals of my dissertation projects were to increase our understanding of metal-mediated associations and improve the utility of metal-controlled dimerization systems.

As a model system, we used the β1 domain of Protein G (Gβ1) as a scaffold to build novel dimer complexes. A published report from a different research group described the generation of a Gβ1 variant that forms a constitutive, symmetric homodimer. This small, well-characterized dimer was an excellent starting model for the design of metal-controlled dimers as considerable biophysical analysis was performed on this variant, and the high resolution three-dimensional structure was solved with X-ray crystallography. We used structure-based rational design to engineer histidine residues at the dimeric interface, which ultimately resulted in high-affinity, metal-controlled protein-protein interactions. Almost all of our design attempts, which contained various interfacial modifications, were shown to form metal-controlled homodimers that bind with moderate to extremely high affinity.

In addition to the successful design of high-affinity symmetric homodimers, we were also interested in generating novel heterodimers that also bind with relative high-affinity. To achieve this we reengineered one of the metal binding sites of a metal-controlled homodimer with oppositely charged side-chains to generate intermolecular protein salt-bridges between an arginine residue on one monomer and two glutamic acid residues on the other. The crystal structures revealed both the designed salt bridges and tetrahedral zinc coordination site. We also demonstrated that binding of the heterodimer complex, which contains the designed salt bridges, was disrupted upon addition of increasing concentrations of sodium chloride. The novel, high-affinity metal-controlled homo- and hetero-dimer proteins could potentially be used as highly effective building blocks for novel biomaterials.