Metal-Mediated Multiprotein Structures for Ester Hydrolysis

We have demonstrated that we can control protein self-assembly by using metal coordination chemistry. Our goal is to design a Zinc-binding site that resembles the active site of Carbonic anhydrase II, with esterase activity. The model protein cytochrome cb₅₆₂ was modified by introducing metal binding histidine residues which allowed the protein to tetramerize in the presence of metal. Further modifications to this model, by using computational interface redesign, gave rise to a stable metal-free tetramer that we can further use as a template for our studies (RIDC-1 96C). This tetramer was stabilized by hydrophobic interactions along one interface and a covalent linkage at the second interface. The resulting metal-free tetramer was elastic along one of the interfaces, which made it difficult to control the coordination environment. Adding a second covalent linkage resulted in a more rigid tetramer (RIDC-1 96C 81C). We have been making mutations at the tetrameric interface in order to try to force a coordination environment with three histidine residues and a water molecule as the ligands. The esterase activites will be measured spectrophotometrically with p-nitrohenyl acetate. Once we achieve esterase activity, we will further characterize the oligomeric structure by using analytical ultracentrifugation, inductively coupled plasma atomic emission spectroscopy and protein crystallography.