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

该提议旨在提高金属蛋白MID 1 sc 10的酯酶活性以达到酶的完美，以及应用合理设计和实验室进化将新的酶活性引入到这种从头设计的肽支架中。将用于本研究的人工肽支架MID 1 sc由两个螺旋-转角-螺旋基序、一个锌配位位点和一个疏水口袋组成。由于支架的高可塑性，有可能将其进化为具有酯酶活性的高活性金属蛋白。最初的MID 1还显示出轻微的磷酸酶活性，这是将这种结构进化为高活性酶的完美起点。此外，使用合理的设计，我们希望将酰胺酶活性作为新的酶功能引入支架中。在第一步中，我们计划交换MID 1支架中的络合金属离子并评估酰胺酶和磷酸酶活性。作为后续步骤，应用集中和随机诱变的实验室进化将用于进一步增强活性并最终产生有效的酶。对于这两个拟议的项目，使用Hilvert实验室中建立的基于荧光的微流体装置，可以对大型DNA文库进行高通量筛选。为了了解酶催化和进化的潜在机制，任何成功进化的酶变体都将使用X射线晶体学和酶动力学测量来表征。

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.