Protein Self-Assembly in Model Microorganisms

描述(申请人提供)：有完整的基因组序列可用， 现在可以检查基因组中的所有蛋白质是否参与其中 在多亚基组件中。不同的蛋白质是如何形成稳定的 从蛋白质结构的角度来看，复合体具有根本的意义 然后折叠起来。此外，识别物理上相互作用的蛋白质可以 为它们的生化和生物功能提供有价值的线索。 绘制蛋白质中负责寡聚化的结构域是一种 结构功能分析的重要部分。此应用程序描述 同时识别和定位低聚结构域的实验 全基因组的规模。 来自酿酒酵母的基因组DNA片段编码的基序可以 自组装将通过一种基于基因融合的遗传方法进行鉴定 方法以大肠埃希菌为宿主。酵母DNA片段文库克隆为 噬菌体Lambda Ci抑制子DNA结合区的基因融合 将接受抑制物活动的选择，这需要组装 变成二聚体或更高级的低聚物。候选基序的初步表征 将利用阻遏系统的独特能力来区分 体内的二聚体和更高级的寡聚形式。虽然精选和 酵母低聚结构域的表征正在进行中， 搜索范围将扩大到寻找两个细菌的自组装结构域，E. 大肠杆菌和结核分枝杆菌，以及两种丝状真菌：粗毛奈氏菌和A. 烟雾剂。尽管这项提议的主要焦点是同型 互动，将开发方法来使用E。 以Coli为基础的双杂交系统用于检测酿酒酵母的蛋白质基序 足以形成异型复合体。 低聚结构域将在大肠杆菌中表达和纯化。大小 排除层析和分析超速离心法将用于 确定它们的齐聚状态。属性域的边界 形成稳定络合物的充要条件将由部分决定 蛋白水解酶，然后用N-末端分析抗蛋白酶片段 多肽测序和质谱分析。可溶性齐聚反应的结构 结构域将由X射线结晶学确定。表达载体将是 利用低聚结构域作为“显性负性”抑制剂 在酿酒酵母和大肠杆菌中。 这项工作将为人类健康提供重要的见解 蛋白质分类学，蛋白质设计材料，遗传学研究的新工具 在模式生物(酿酒酵母和大肠杆菌)和重要的人类病原体中 (结核分枝杆菌和烟曲霉菌)，以及基于 蛋白质之间的相互作用。

DESCRIPTION (provided by applicant): With complete genome sequences available, it is now possible to examine all of the proteins in a genome for involvement in multisubunit assemblies. How different proteins are able to form stable complexes is of fundamental interest from the perspective of protein structure and folding. In addition, identifying proteins that physically interact can provide valuable clues about their biochemical and biological functions. Mapping domains within proteins that are responsible for oligomerization is an important part of structure-function analysis. This application describes experiments to simultaneously identify and localize oligomerization domains on a genome-wide scale. Genomic DNA fragments from S. cerevisiae that encode motifs that can self-assemble will be identified by a genetic approach based on gene fusion methods using E. coli as a host. Libraries of yeast DNA fragments cloned as gene fusions to the DNA binding domain of bacteriophage lambda cI repressor will be subjected to selection for repressor activity, which requires assembly into dimers or higher oligomers. Initial characterization of candidate motifs will exploit the unique ability of the repressor system to distinguish between dimers and higher oligomeric forms in vivo. While the selection and characterization of oligomerization domains from yeast is in progress, the search will be extended to find self-assembling domains from two bacteria, E. coli and M. tuberculosis, and two filamentous fungi, N. crassa and A. fumigatus. Although the primary focus of this proposal is on homotypic interactions, methods will be developed to use combinations of libraries in E. coli-based two-hybrid systems to examine protein motifs from S. cerevisiae that are sufficient to form heterotypic complexes. Oligomerization domains will be expressed and purified from E. coli. Size exclusion chromatography and analytical ultracentrifugation will be used to determine their oligomerization states. The boundaries of the domains that are necessary and sufficient to form stable complexes will be determined by partial proteolysis, followed by analysis of protease resistant fragments by N-terminal peptide sequencing and mass spectrometry. Structures of soluble oligomerization domains will be determined by X-ray crystallography. Expression vectors will be developed to use the oligomerization domains as "dominant negative" inhibitors in S. cerevisiae and in E. coli. This work will contribute to human health by providing important insights into protein taxonomy, materials for protein design, new tools for genetic studies in model organisms (S. cerevisiae and E. coli) and important human pathogens (M. tuberculosis and A. fumigatus), and new drug targets based on protein-protein interactions.