Elucidating the folding mechanism of a multi domain protein by combining quench-flow hydrogen exchange methodology with multidimensional NMR spectroscopy

通过结合骤冷流氢交换方法与多维核磁共振波谱阐明多结构域蛋白质的折叠机制

• 批准号: 501283364
• 负责人: Professor Dr. Michael Kovermann
• 金额: --
• 依托单位: Arbeitsgruppe Magnetische Resonanzspektroskopie (NMR)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/501283364?language=en
• 关键词: Elucidating; folding; mechanism; multi; domain

Elucidating the folding mechanism of a protein represents one major goal in structural biology. The majority of experimental studies aiming to unravel the folding path of a protein have focused on single domain proteins so far. The fact that about 65 % of the procaryotic and about 80 % of the eucaryotic proteome comprise multi domain proteins corroborates the distinct interest in obtaining spatial- and time-dependent insights into the folding path of proteins possessing higher complexity. Here, a synergistic combination between quench-flow hydrogen exchange and multidimensional NMR spectroscopy will be applied to three domain Adenylate Kinase (AdK) to determine the folding process of this enzyme on a millisecond to second time scale at atomic resolution. Functionally, the catalytic activity of native AdK ensures the maintenance of cellular concentration of adenylate nucleotides and acts for this reason as an eminent model system to study the precise relation between folding and function. The proposed work will include natural ligands and an enzymatic inhibitor in the experimental setup enabling to probe binding features within the folding landscape of AdK. Thus, this project aims to make new contributions regarding the following questions: Does AdK refold in a domain dependent manner? Which lifetime do potential folding intermediates possess? How much native structure is needed to act as a functional enzyme? How does the formation of secondary and tertiary structure accounts for functionality of an enzyme? It can be assumed that the precise knowledge of the folding mechanism of three domain AdK in space and time prepares an excellent ground for a better understanding of folding and function of multi domain proteins in general as well as demanding applications e.g. in protein design can be envisioned.

阐明蛋白质的折叠机制是结构生物学的一个主要目标。迄今为止，大多数旨在揭示蛋白质折叠路径的实验研究都集中在单结构域蛋白质上。约65%的原核蛋白质组和约80%的真核蛋白质组包含多结构域蛋白质的事实证实了对获得对具有更高复杂性的蛋白质的折叠路径的空间和时间依赖性见解的独特兴趣。在这里，猝灭流氢交换和多维NMR光谱之间的协同组合将被应用于三个域腺苷酸激酶（AdK），以确定这种酶的折叠过程在毫秒到秒的时间尺度在原子分辨率。在功能上，天然AdK的催化活性确保了腺苷酸核苷酸的细胞浓度的维持，并因此作为研究折叠和功能之间精确关系的杰出模型系统。拟议的工作将包括天然配体和酶抑制剂的实验设置，使探针内的AdK折叠景观的结合功能。因此，该项目旨在对以下问题做出新的贡献：AdK是否以域依赖的方式重新折叠？潜在的折叠中间体具有什么样的寿命？需要多少天然结构才能作为一种功能性酶发挥作用？二级和三级结构的形成如何解释酶的功能？可以认为，三结构域AdK在空间和时间上的折叠机制的精确知识为更好地理解多结构域蛋白质的折叠和功能以及例如在蛋白质设计中的要求苛刻的应用提供了良好的基础。