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

 描述（由申请人提供）：病原菌必须通过胃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，有助于在细菌致病之前杀死它们。然而，在一些最具感染性的细菌中，ATP非依赖性伴侣蛋白HdeA在帮助细菌在低pH下存活方面起主要作用。HdeA的作用机制相当独特，因为它是处于其活化状态的未折叠单体蛋白。它的工作是保护其他蛋白质免于错误折叠和聚集，因为细胞通过胃的恶劣环境过渡到肠的中性环境。一旦细菌进入肠道，HdeA释放这些蛋白质并重新折叠成其非活性二聚体构象。生物物理学研究提供了线索，HdeA展开低于pH 3.0，并与其结合伙伴使用在折叠蛋白质的二聚体界面发现的疏水残基相互作用。然而，缺乏详细监测在低于3.0的多个pH值下的单体化、解折叠和活化机制的数据。此外，通常还不能很好地理解非线性无序蛋白质的性质。 具体目标。我们建议追求一个彻底的，原子级的调查HdeA在低pH值的激活机制，使用核磁共振（NMR）光谱作为我们的主要分析工具。由于它是可能的，细胞拥挤是一个重要的贡献者或理解的HdeA活性（特别是在其未折叠状态），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，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中伴侣活性活化的特定结构和动态变化; 2）研究与体外HdeA相比，在pH 6.0和2.0之间细胞内或裂解物中HdeA发生的结构和动态变化的差异。NMR实验将包括滴定监测化学位移变化作为pH值的函数，氢交换和3D实验，以结构表征HdeA，和自旋弛豫实验，以分析HdeA中的主链和侧链蛋白质运动在多个时间尺度和多个pH值。 健康相关的意义。由致病菌引起的肠道感染引起的痢疾每年在全世界造成100多万人死亡。如果我们能够理解HdeA如何感知pH值变化并被pH值变化触发，我们就可以更好地理解这种类型的酸应激分子伴侣如何帮助细菌在极端条件下生存。有了这一认识，我们将能够提高疫苗或其他疗法的靶向，使HdeA的活性丧失，从而削弱这些致病菌的感染性。

 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.