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

本课题旨在通过合理设计和实验室进化，提高金属蛋白MID1sc10的酯酶活性，达到酶促完善的目的，并将新的酶促活性引入到这个从头设计的肽支架中。本研究中使用的人工肽支架MID1sc由两个螺旋-转-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，它有可能进化成具有酯酶活性的高活性金属蛋白。最初的MID1也显示出轻微的磷酸酶活性，这是将该结构进化为高活性酶的完美起点。此外，通过合理的设计，我们希望将酰胺酶活性作为一种新的酶功能引入支架。在第一步，我们计划交换MID1支架中的络合金属离子，并评估酰胺酶和磷酸酶的活性。作为后续步骤，实验室进化应用集中和随机诱变将用于进一步提高活性，并最终产生有效的酶。对于这两个拟议的项目，使用希尔弗特实验室建立的基于荧光的微流体装置，允许对大型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.