打结蛋白YibK去折叠过程中的动态变化及其中间态构象的单分子研究

Knotted proteins play an important role in cell signal transduction, DNA/RNA metabolic regulation. However, until now the formation and its biological meaning of knots, together with their dynamics and unfolding mechanism during denaturation process were kept unknown. These brought us a new kind of protein folding problem.. To address this issue, the trifoil-knotted methyltransferase, YibK from Haemophilus influenza, will be studied as a model knotted protein. Biophysical methods, such as time-resolved fluorescence techniques, single molecular fluorescence techniques, single molecular force spectroscopy and molecular simulation techniques, were employed to study the dynamics and unfolding mechanism of YibK during denaturation. The conformation transition and its kinetics of YibK knotted region with different concentration of denaturant will be determined to provide detailed information on the YibK unfolding process, and the intermediate states of YibK during unfolding process will also be characterized to illustrate the detailed pathway and molecular mechanism of YibK unfolding process. This research will not only shed light on the unfolding mechanism of knotted proteins and strengthen our knowledge in protein folding mechanisms, but also provide important information on treatment of related disease and drug design.

打结蛋白在细胞的信号传递、遗传物质代谢调控等生理过程中发挥着重要作用。然而，目前对于打结蛋白中拓扑异构结的形成机理及其生物学意义，以及变性条件下打结蛋白去折叠过程的动态变化及其去折叠机制仍不十分清楚，由此带来了一类全新的蛋白折叠问题。 .本项目拟以细菌甲基转移酶YibK为研究对象，综合利用时间分辨荧光技术、单分子荧光技术、单分子力谱技术以及计算机模拟技术等多种生物物理化学技术，对YibK在变性条件下的去折叠过程进行研究。通过测定不同浓度变性剂作用下YibK去折叠过程的构象变化及其动力学参数，为YibK去折叠过程的动态变化提供详细信息；同时通过对YibK去折叠过程中间态构象的表征，揭示YibK的去折叠途径及其分子机制。本项目的实施不仅可以深入理解蛋白质中拓扑异构结的折叠过程及其生物学意义，并丰富蛋白质折叠机理提供重要信息，还可以为相关的疾病治疗及药物设计奠定理论基础。

打结蛋白在细胞信号传递、代谢调控等生理过程中发挥着重要作用。目前对于打结蛋白中拓扑异构结的折叠机理及其生物学意义仍不清楚。本项目以1O6D、YibK和YbeA三种打结蛋白为研究对象，综合利用时间分辨荧光、单分子荧光及计算机模拟等多种生物物理化学技术，对打结蛋白在变性条件下的去折叠过程进行研究。结果发现，打结蛋白在变性剂作用下，会发生去折叠过程，该过程中打结区域发生明显构象变化，打结区域逐渐向C末端滑动，且拓扑异构结变大、变松散。单分子荧光研究表明，打结蛋白去折叠过程中存在明显的中间态构象，这为详细阐明打结蛋白的去折叠过程奠定重要的工作基础。分子动力学模拟结果发现，打结结构对于稳定打结蛋白的稳定性和二聚化结构具有重要作用，从而为打结蛋白发挥其生物学功能提供了重要的结构保障。本项目的实施为深入理解蛋白质中拓扑异构结的折叠过程及其生物学意义，以及打结蛋白相关疾病治疗及药物设计奠定理论基础。