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

项目摘要/摘要 背景资料。病原菌必须在胃部的高酸性环境中传播才能 它们可以到达并感染肠道。因此，胃是一个重要的屏障，有助于杀死 许多细菌才能致病。然而，在一些传染性最强的细菌中，ATP- 独立的分子伴侣HdeA和HdeB在帮助细菌在低pH条件下存活方面发挥了重要作用。他们的工作是 当细胞在恶劣的环境中过渡时，保护其他蛋白质不会错误折叠和聚集 进入胃部和肠道的中性环境。HdeB在中等pH值时是活跃的，而 HdeA在胃酸典型的低pH条件下起作用。尽管有各种模型可用来解释 关于这两个伴侣之间的相互作用，目前还不清楚哪一个是正确的，如果有的话。 明确的目标。这项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。 对两个伴侣蛋白表面上的协同作用机制进行深入研究 蛋白质的作用，并探索特定残基的作用，触发或修改HdeA或 HdeB。目标1是检查HdeA和HdeB作为一个功能与监护人客户的角色和交互 酸碱度。同位素标记和核磁共振光谱的独特性质将被用来监测每一个 HdeA、HdeB和客户蛋白混合物中的单独蛋白质，从而提供不同的优势 积分获取前所未有的细节。第二个目标是探索HdeA中伴侣蛋白的激活和稳定部位 和HdeB使用靶向突变。在这里，将使用各种技术来密切评估 每种蛋白质都与功能和/或激活中的关键角色有关，包括 HdeB的二聚体界面和HdeA的二硫键有助于维持半折叠结构 被认为对它的伴侣功能很重要。 与健康相关的意义。由病原体引起的肠道感染引起的痢疾，至少会导致死亡 全球每年有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伴侣如何帮助细菌在极端条件下生存。改进 了解这一点可以让研究人员设计疫苗或其他疗法来阻止这种活动 HdeA和HdeB的感染，从而减弱这些致病菌的感染力。

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.