In vitro and in-cell investigation of the acid-stress chaperone HdeA

 描述(申请人提供)：病原菌必须穿过胃部的高酸性环境，才能到达并感染肠道。因此，胃是一个重要的屏障，它有助于在许多细菌致病之前杀死它们。然而，在一些最具感染性的细菌中，不依赖于ATP的伴侣HdeA在帮助细菌在低pH下生存方面发挥了重要作用。HdeA的作用机制相当独特，因为它是一种处于激活状态的未折叠的单体蛋白。它的工作是保护其他蛋白质在细胞通过胃的恶劣环境进入肠道的中性环境时不会错误折叠和聚集。一旦细菌进入肠道，HdeA就会释放这些蛋白质，并重新折叠成不活跃的二聚体构象。生物物理研究提供了线索，即HdeA在pH 3.0以下展开，并利用在折叠蛋白质的二聚体界面上发现的疏水残基与其结合伙伴相互作用。然而，在多个pH值低于3.0的情况下，缺乏详细监测单体、去折叠和活化机制的数据。此外，无序蛋白质的性质通常还没有被很好地理解。 明确的目标。我们建议使用核磁共振光谱作为我们的主要分析工具，对低pH下HdeA的激活机制进行彻底的、原子水平的研究。由于细胞拥挤可能是对HdeA活性(特别是在其未折叠状态下)的重要贡献或理解，我们建议在体外和细胞内研究HdeA激活的机制。HdeA也将是一个很好的模型系统，可以提高我们对内在无序蛋白质功能的理解。我们的具体目标是：1)确定在pH 3.0和2.0之间触发体外HdeA中伴侣活性激活的特定结构和动态变化；2)研究在pH 6.0和2.0之间细胞内或裂解物中与体外HdeA相比所发生的结构和动态变化的差异。核磁共振实验将包括监测作为pH函数的化学位移变化的滴定，用于表征HdeA结构的3D实验，以及用于分析HdeA在多个时间尺度和多个pH值下的主链和侧链蛋白质运动的自旋松弛实验。 与健康相关的意义。痢疾是由病原菌引起的肠道感染引起的，全世界每年有100多万人死于痢疾。如果我们能够理解HdeA是如何感知并被pH变化触发的，我们就可以更好地理解这种类型的酸应激伴侣如何帮助细菌在极端条件下生存。有了这一认识，我们将能够改进针对疫苗或其他治疗方法的靶向，这些疫苗或治疗方法可以使HdeA失去活性，从而削弱这些病原菌的传染性。

 DESCRIPTION (provided by applicant): Pathogenic bacteria must travel through the highly acidic environment of the stomach before they can reach and infect the intestines. The stomach is therefore an important barricade which helps to kill many bacteria before they can cause illness. In some of the most infectious bacteria, however, the ATP- independent chaperone HdeA plays a major role in aiding bacterial survival at low pH. HdeA's mechanism of action is rather unique, in that it is an unfolded monomeric protein in its activated state. Its job is to protect other proteins from misfolding and aggregating as the cell transitions through the harsh environment of the stomach and into the neutral environment of the intestines. Once the bacteria enter the intestinal tract, HdeA releases these proteins and refolds into its inactive dimer conformation. Biophysical studies have provided clues that HdeA unfolds below pH 3.0 and interacts with its binding partners using hydrophobic residues found at the dimer interface of the folded protein. However, there is a dearth of data that monitors, in detail, the mechanism of monomerization, unfolding and activation at multiple pH values below 3.0. In addition, the properties of instrinsically disordered proteins are generally not well-understood. Specific aims. We propose to pursue a thorough, atomic-level investigation of the mechanism of activation of HdeA at low pH, using Nuclear Magnetic Resonance (NMR) spectroscopy as our primary analytical tool. Since it is likely that cellular crowding is an important contributor to or understanding of HdeA activity (especially in its unfolded state) we propose to study the mechanism of HdeA activation both in vitro and in-cell. HdeA will also be an excellent model system to improve our understanding of functionality in an intrinsically disordered protein. Our specific aims are to 1) determine the specific structural and dynamic changes that trigger activation of chaperone activities in HdeA in vitro between pH 3.0 and 2.0 and 2) investigate the differences in structural and dynamic changes that occur in HdeA in-cell or in lysate between pH 6.0 and 2.0 compared to HdeA in vitro. NMR experiments will include titrations to monitor chemical shift changes as a function of pH, hydrogen exchange and 3D experiments to structurally characterize HdeA, and spin relaxation experiments to analyze backbone and side chain protein motions in HdeA at multiple timescales and multiple pH values. Health-related significance. Dysentery, caused by intestinal infection by pathogenic bacteria, kills over one million people per year worldwide. If we can understand how HdeA senses and is triggered by pH changes, we can better understand how this type of acid-stress chaperone helps bacteria survive under extreme conditions. Armed with this understanding we will be able to improve targeting for vaccines or other therapeutics that can disable the activities of HdeA and thereby weaken the infectivity of these pathogenic bacteria.