Reaching enzymatic perfection of the de novo designed metalloprotein MID1sc10 and implementing of new enzymatic activities into a flexible protein scaffold by exchanging metal ions and directed evolution

点击翻译按钮获取中文摘要

This proposal aims at improving the esterase activity of the metalloprotein MID1sc10 to reach enzymatic perfection as well as introducing novel enzymatic activities into this de novo designed peptide scaffold applying rational design and laboratory evolution. The artificial peptide scaffold MID1sc that will be used in this study consists of two helix-turn-helix motifs, a zinc coordination site and a hydrophobic pocket. Because of the high plasticity of the scaffold, it was possible to evolve it to a highly active metalloprotein with esterase activity. The initial MID1 also showed a slight phosphatase activity, a perfect starting point to evolve this structure to a highly active enzyme. Moreover, using rational design, we want to introduce amidase activity as a novel enzymatic functionality into the scaffold. In a first step, we plan to exchange the complexed metal ion in the MID1 scaffold and evaluate the amidase and phosphatase activity. As subsequent steps, laboratory evolution applying focused and random mutagenesis will be used to further enhance the activities and ultimately yield an efficient enzyme. For both proposed projects, the use of an established fluorescence-based microfluidics setup in the Hilvert lab that allows high-throughput screening of large DNA libraries. In order to understand the underlying mechanisms of enzyme catalysis and evolution, any successfully evolved enzyme variant will be characterized using X-ray crystallography as well as enzyme kinetics measurements.