NMR STUDIES OF PROTEIN-PROTEIN RECOGNITION IN BLOOD CELL DEVELOPMENT

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。该项目的总体目标是通过核磁共振（NMR）光谱表征参与III型分泌的细菌针蛋白的结构和动力学。许多引起重要人类疾病的细菌需要III型分泌系统才能感染宿主。III型分泌物的一个突出特征是管状结构，即针状器，其由单一类型的针蛋白聚合而成。 针装置在细菌和宿主细胞之间形成物理接触，并具有25 º的中央通道，用于将细菌蛋白毒素转运到宿主细胞中。我们已经表达和纯化的单体形式的细菌针蛋白从类鼻疽伯克霍尔德氏菌和鼠伤寒沙门氏菌，BsaL和PrgI，分别，并已获得高质量的NMR谱。我们的目标是表征BsaL和PrgI的NMR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NMR结构，并表明它是由一个双螺旋束两侧的残基是在部分螺旋构象。我们还希望在今年完成PrgI的NMR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，并评估它们在针装置组装中的作用，并将这些数据与细菌发病机制相关联。 我们对BsaL的研究首次报道了任何III型针蛋白的原子水平结构，并为进一步的NMR研究提供了基础，这些研究旨在表征针蛋白的结构和动力学，它们的蛋白质-蛋白质相互作用及其在发病机制中的作用。我们希望提供这些蛋白质如何在原子水平上工作的细节，并且这些知识可用于设计针对破坏III型分泌系统的新型抗菌疗法。

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.