NMR STUDIES OF PROTEIN-PROTEIN RECOGNITION IN BLOOD CELL DEVELOPMENT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目的总体目标是通过核磁共振(NMR)光谱表征参与III型分泌物的细菌针蛋白质的结构和动力学。许多导致人类重大疾病的细菌需要III型分泌系统才能感染宿主。III型分泌物的一个显著特征是管状结构，即针状结构，它是由单一类型的针状蛋白聚合而成的。针头装置在细菌和宿主细胞之间形成物理接触，并有一个25？的中央通道，用于将细菌蛋白毒素输送到宿主细胞。我们分别从伯克霍尔德氏菌和鼠伤寒沙门氏菌Bsal和PrgI中表达和纯化了细菌针蛋白的单体形式，并获得了高质量的核磁共振谱。我们的目标是描述Bsal和PrgI的核磁共振结构和动力学，并将这些数据与发病机制相关联。我们已经完成了Bsal的核磁共振结构，并表明它由一个两螺旋束组成，两侧是部分螺旋构象的残基。我们还预计今年完成PrgI的核磁共振结构。我们将使用核磁共振骨架动力学来表征这些蛋白质的运动，并评估它们在针具组装中的作用，并将这些数据与细菌发病机制联系起来。我们在BSAL上的工作首次报道了任何III型针状蛋白的原子水平结构，并为进一步的核磁共振研究奠定了基础，目的是表征针状蛋白的结构和动力学，它们的蛋白质-蛋白质相互作用，以及它们在发病机制中的作用。我们希望提供这些蛋白质在原子水平上如何工作的细节，这些知识可以用来设计新的抗菌疗法，目的是扰乱III型分泌系统。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project is to characterize the structures and dynamics of bacterial needle proteins involved in the type III secretion by nuclear magnetic resonance (NMR) spectroscopy. Many bacteria that cause important human diseases require a type III secretion system in order to infect their hosts. A prominent feature of the type III secretion is a tube-like structure, the needle apparatus, which is assembled from the polymerization of a single type of needle protein. The needle apparatus forms a physical contact between the bacterium and the host cell, and has a central channel of 25 ¿ that is used to transit bacterial protein toxins into the host cell. We have expressed and purified the monomeric forms of the bacterial needle proteins from Burkholderia pseudomallei and Salmonella typhimurium, BsaL and PrgI, respectively, and have acquired high quality NMR spectra. Our objective is to characterize the NMR structures and dynamics of BsaL and PrgI, and correlate these data to pathogenesis. We have completed the NMR structure of BsaL and showed that it is composed of a two-helix bundle flanked by residues that are in partial helical conformation. We also expect to complete the NMR structure of PrgI this year. We will use NMR backbone dynamics to characterize the motions of these proteins, and assess their roles in the assembly of the needle apparatus, and correlate these data with bacterial pathogenesis. Our work on BsaL represents the first report of an atomic level structure for any type III needle protein, and provides the foundation for further NMR studies aimed at characterizing the structures and dynamics of needle proteins, their protein-protein interactions, and their roles in pathogenesis. We hope to provide the details of how these proteins work at the atomic level, and that knowledge can be used to design novel antibacterial therapy targeted at disrupting the type III secretion system.