Protein Self-Assembly in Model Microorganisms

模型微生物中的蛋白质自组装

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/6630431
• 关键词: Escherichia coli; Saccharomyces cerevisiae; bacterial genetics; bacterial proteins; bacteriophage lambda; chimeric proteins; crystallization; expression cloning; functional /structural genomics; fungal proteins; genetic library; intermolecular interaction; microorganism; model design /development; molecular assembly /self assembly; molecular biology information system; molecular site; physical model; polymerization; protein purification; protein sequence; protein structure function; proteomics; structural biology; yeast two hybrid system

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）：随着完整基因组序列的可用， 现在可以检测基因组中所有的蛋白质 在多子单元组件中。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，复合物具有根本的意义 和折叠。此外，识别物理相互作用的蛋白质可以 提供了有关其生化和生物功能的有价值的线索。 在蛋白质中定位负责寡聚化的结构域是一种新的方法。 结构功能分析的重要组成部分。本申请描述 同时识别和定位寡聚化结构域的实验 a genome-wide基因组scale规模. S.酿酒酵母基因编码的基序， 自组装将通过基于基因融合的遗传方法来鉴定 方法采用E.大肠杆菌作为宿主。克隆的酵母DNA片段文库， 与λ噬菌体cI阻遏物DNA结合域的基因融合 将受到阻遏物活性的选择，这需要组装 转化为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 二聚体和更高级的低聚体形式。虽然选择和 酵母寡聚化结构域的表征正在进行中， 搜索将扩展到从两种细菌中找到自组装结构域，E。 coli和M. tuberculosis和两种丝状真菌N. crassa和A. 烟熏。虽然这项建议的主要重点是在同型 相互作用，将开发方法来使用E. 大肠杆菌为基础的双杂交系统，以检查蛋白基序从S。酿酒， 足以形成异型复合物。 寡聚化结构域将从E.杆菌大小 将使用排阻色谱法和分析超速离心法来 确定它们的低聚状态。域的边界， 形成稳定复合物的必要和充分条件将由部分 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。可溶性低聚反应的结构 域将通过X射线晶体学确定。表达载体将是 开发使用寡聚化结构域作为“显性负性”抑制剂 in S. cerevisiae和E.杆菌 这项工作将有助于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 在模式生物中（S. cerevisiae和E.大肠杆菌）和重要的人类病原体 （M.结核病和A.烟曲霉），以及基于 蛋白质相互作用