Chemoselective DNA conjugation of proteins to study multienzyme clomplexes

这个项目涉及到一个通用平台的开发，以产生对生物系统询问有用的多功能蛋白质结构。该平台技术是基于化学选择性蛋白质连接方法和结构DNA纳米技术的结合。特别是，我们将建立来自多模聚酮合成酶(PKS)的重组蛋白与DNA或PNA寡核苷酸的化学酶连接，这些DNA或PNA寡核苷酸被短肽标记衍生，作为细菌转肽酶Sortase的连接底物。由此产生的DNA/PNA标记的PKS模块将通过Watson-Crick碱基配对在均相溶液中组装，特别是在DNA折纸技术产生的二维核酸支架的表面上。在高度模块化的方法中，将构建超分子多酶复合体，其中PKS结构域的空间构型由纳米级的DNA支架控制。一方面，该项目将有助于详细了解多酶复合体的空间组织、构象灵活性和其他基本原理。另一方面，这里开发的平台将使人们能够方便地访问大量空间上定义良好的多功能蛋白质复合体，用于模拟、操作和分析涉及基因调控、信号转导、分裂或其他细胞功能和特征的自然发生的超分子组合。

This project concerns the development of a generic platform to generate multfunctional protein constructs useful for interrogation of biological systems. The platform technology is based on the combination of chemoselective protein ligation methods and structural DNA nanotechnology. In particular, we will establish the chemoenzymatic ligation of recombinant proteins derived from multimodular Polyketide Synthases (PKS) with DNA or PNA oligonucleotides, which are derivatized with a short peptide tag, serving as the ligation substrate of bacterial transpeptidase Sortase. The resulting DNA-/PNA-tagged PKS modules will be assembled by Watson-Crick base pairing in homogeneous solution and, in particular, on the surface of two-dimensional nucleic acid scaffolds generated by the DNA origami technique. In a highly modular approach, supramolecular multienzyme complexes will be built, in which the spatial configuration of the PKS domains is controlled by the DNA scaffold with nanometer precision. On the one hand, this project will contribute to the detailed understanding of the spatial organization, conformational flexibility and other fundamental principles governing multienzyme complexes. On the other hand, the platform developed here will enable ready access to numerous spatially well-defined multifunctional protein complexes for mimicking, manipulation and analysis of natural occuring supramolecular assemblies involved in gene regulation, signal transduction, division, or other cellular function and traits.