Protein Self-Assembly in Model Microorganisms

模型微生物中的蛋白质自组装

基本信息

批准号：
6630431
负责人：
JAMES C HU
金额：
$ 26.87万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2005-07-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：具有完整的基因组序列，现在可以检查基因组中的所有蛋白质是否参与在多亚基组装体中。不同的蛋白质如何形成稳定的从蛋白质结构的角度来看，复合物具有根本意义和折叠。此外，识别物理相互作用的蛋白质可以提供有关其生化和生物学功能的宝贵线索。映射蛋白质内负责寡聚化的结构域是一个结构功能分析的重要组成部分。该应用程序描述了同时识别和定位寡聚结构域的实验全基因组规模。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基于基因融合的遗传方法进行鉴定使用大肠杆菌作为宿主的方法。酵母 DNA 片段文库克隆为与噬菌体 lambda cI 阻遏物的 DNA 结合域的基因融合将受到抑制活性的选择，这需要组装成二聚体或更高级的寡聚体。候选主题的初步表征将利用阻遏系统的独特能力来区分体内二聚体和更高的寡聚形式。在选择和酵母寡聚化结构域的表征正在进行中，搜索范围将扩大到寻找两种细菌的自组装结构域，即大肠杆菌。大肠杆菌和结核分枝杆菌，以及两种丝状真菌，粗糙链球菌和A. 熏烟。尽管该提案的主要焦点是同型相互作用，将开发方法来使用大肠杆菌中的库组合。基于大肠杆菌的双杂交系统来检查酿酒酵母的蛋白质基序足以形成异型复合物。寡聚化结构域将从大肠杆菌中表达和纯化。尺寸将使用排阻色谱法和分析超速离心法确定它们的低聚状态。域的边界是形成稳定复合物的必要和充分条件将由部分决定蛋白水解，然后通过 N 末端分析蛋白酶抗性片段肽测序和质谱分析。可溶性低聚结构域将通过X射线晶体学来确定。表达载体将是开发使用寡聚化结构域作为“显性失活”抑制剂在酿酒酵母和大肠杆菌中。这项工作将通过提供重要的见解来为人类健康做出贡献蛋白质分类学、蛋白质设计材料、遗传研究新工具存在于模式生物（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），以及基于蛋白质-蛋白质相互作用。