Synergy between acid stress chaperones HdeA and HdeB with clients and their key sites of activity

项目概要/摘要 背景致病细菌必须在胃的高酸性环境中传播， 它们可以到达并感染肠道。因此，胃是一个重要的屏障， 许多细菌在它们致病之前。然而，在一些最具感染性的细菌中，ATP- 独立的分子伴侣HdeA和HdeB在帮助细菌在低pH下存活方面起主要作用。它们的工作是 保护其他蛋白质免于在细胞过渡到恶劣的环境时发生错误折叠和聚集， 进入肠道的中性环境。HdeB在中等pH值下具有活性，而 HdeA在胃酸典型的低pH下发挥作用。虽然有各种模型可以解释 这两个伴侣之间的相互作用，目前还不清楚，如果有的话，是正确的。 具体目标。拟议工作的目标是使用核磁共振波谱和其他生物物理技术 深入研究这两种分子伴侣的协同作用机制， 蛋白质的作用，并探测触发或修饰HdeA或 HdeB。目标#1是检查HdeA和HdeB与伴侣客户端的角色和交互作用 同位素标记和NMR光谱学的独特性质将用于监测每一个 在HdeA、HdeB和客户蛋白的混合物中单独地添加蛋白，从而提供不同的Vantage 以获得前所未有的细节。目的#2是探测HdeA中伴侣蛋白激活和稳定性的位点 和HdeB。在这里，各种技术将被用来密切评估的部分， 每种蛋白质都与功能和/或激活中的重要作用有关，包括在 HdeB的二聚体界面和HdeA中的二硫键，这有助于维持半折叠结构 被认为对它的伴侣蛋白功能很重要。 健康相关的意义。痢疾，由致病菌引起的肠道感染， 全球每年35万人。如果我们能够阐明HdeA和HdeB在 客户蛋白的存在，以及特定残基或区域的机械重要性，我们可以 更好地了解这些酸应激分子伴侣如何帮助细菌在极端条件下生存。改进 这种了解可以为研究人员设计疫苗或其他治疗方法提供信息， 的HdeA和HdeB，从而削弱这些病原菌的感染性。

PROJECT SUMMARY / ABSTRACT Background. 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, ATP- independent chaperones HdeA and HdeB play major roles in aiding bacterial survival at low pH. Their 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. HdeB is active at intermediate pH values, while HdeA functions at the low pH typical of stomach acid. Although there are various models available to explain the interplay between the two chaperones, it is still unclear which, if any, is correct. Specific aims. The goal of the proposed work is to use NMR spectroscopy and other biophysical techniques to pursue an in-depth investigation of the apparently synergistic mechanism by which the two chaperone proteins operate and to probe the roles of specific residues that trigger or modify the activation of HdeA or HdeB. Aim #1 is to examine the roles and interactions of HdeA and HdeB with chaperone clients as a function of pH. Isotopic labeling and the unique properties of NMR spectroscopy will be employed to monitor each protein individually within a mixture of HdeA, HdeB and a client protein, thereby providing different vantage points to obtain unprecedented detail. Aim #2 is to probe sites of chaperone activation and stability in HdeA and HdeB using targeted mutations. Here, a variety of techniques will be used to closely assess segments of each protein that have been linked to essential roles in function and/or activation, including a key tryptophan in the dimer interface of HdeB and the disulfide bond in HdeA, which helps to maintain the semi-folded structure believed to be important to its chaperone function. Health-related significance. Dysentery, caused by intestinal infection by pathogenic bacteria, kills at least 350,000 people per year worldwide. If we can elucidate the individual and collective roles of HdeA and HdeB in the presence of client proteins, as well as the mechanistic importance of specific residues or regions, we can better understand how these acid-stress chaperones help bacteria survive under extreme conditions. Improved understanding can inform researchers designing vaccines or other therapeutics that can disable the activities of HdeA and HdeB and thereby weaken the infectivity of these pathogenic bacteria.