Design and Construction of Synthetic Metal-Binding Proteins

摘要

The goal of this project is to assemble a systematic framework for design and construction of synthetic metal-binding proteins for metal recognition and recovery. In the first phase of this work, we are investigating metal-binding sites that can be built into the surface of a protein with relatively few amino acid substitutions. One such site, consisting of two histidines positioned His-X-X-X-His in an alpha-helix, has been built into yeast iso-1-cytochrome c. The resulting mutant protein binds Cu(II) and other metals with high affinity.In addition to the protein engineering work, a novel technique for quantifying metal-protein interactions has been developed. This technique is based on the partitioning of metal-binding proteins between two aqueous-polymer phases. When polymer in one phase is derivatized with the appropriate metal chelate (e.g. poly(ethylene glycol)-IDA-Cu(II), partitioning of a protein between the two phases depends on the number of binding sites and the affinity of each site for the metal. We have demonstrated that metal-protein binding constants can be obtained for binding sites accessible to the chelated metals. Keywords: Protein-metal interaction: Binding sites. (kr)