Structural Studies of NAGS

NAGS的结构研究

• 批准号: 6808170
• 负责人: DASHUANG SHI
• 金额: $ 12.47万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6808170
• 关键词: Escherichia coli; Mammalia; X ray crystallography; active sites; acyltransferase; aminoacid biosynthesis; ammonia; arginine; blood chemistry; chemical aggregate; chemical stability; chimeric proteins; crystallization; enzyme mechanism; enzyme structure; genetic disorder; genetic manipulation; genetic screening; green fluorescent proteins; maltose; method development; mutant; postdoctoral investigator; protein structure function; solubility; structural biology; urea cycle

DESCRIPTION (provided by applicant): This research training application is aimed to provide both didactic and research training experience to allow the candidate to become an independent investigator. The purpose of training is to expand the expertise of the candidate in molecular biology and molecular microbiology which will complement his knowledge and experience in crystallography. Thus, three of the mentors have expertise in molecular biology and the other two will provide expertise in various aspects of structural analysis of proteins. The research projects aim to understand the correlations between three-dimensional structure and function of N-acetylglutamate synthase (NAGS) encoded by the most recently cloned gene of this unique urea cycle enzyme, thought to be involved in the regulation of ureagenesis. Very little is known on the structure of any NAGS protein. The NAGS genes of bacteria, fungi and mammals are much more diverse than other arginine-biosynthesis and urea-cycle genes. It is very interesting, therefore, to explore at the protein level whether the three-dimensional structures are also very different and how the enzymes respond differently to L-arginine. A strategy to overcome the insolubility and aggregation of mammalian and E. coil NAGS is proposed. This includes using random mutagenesis of the NAGS gene followed by GFP screening and functional assay to identify the highly soluble and stable mutants. The second methodology uses the fusion of NAGS to a soluble protein such as green fluorescent protein (GFP) or maltose binding protein (MBP) to increase protein solubility and stability. Different length of amino acid linkers with different properties will be produced to examine how they affect the crystallization behavior. The ultimate goal is to study the three-dimensional structures of NAGS enzymes from both lower and higher organisms and to understand their catalytic and regulatory mechanisms, and relationship to inherited defects causing hyperammonaemia.

描述（由申请人提供）： 这个研究培训应用程序的目的是提供教学和研究培训的经验，让候选人成为一个独立的调查员。培训的目的是扩大候选人在分子生物学和分子微生物学方面的专业知识，这将补充他在晶体学方面的知识和经验。因此，其中三名导师具有分子生物学方面的专业知识，另外两名将提供蛋白质结构分析各个方面的专业知识。该研究项目旨在了解N-乙酰谷氨酸合酶（NAGS）的三维结构和功能之间的相关性，该酶由最近克隆的这种独特的尿素循环酶基因编码，被认为参与了尿素生成的调节。人们对任何NAGS蛋白的结构知之甚少。细菌、真菌和哺乳动物的NAGS基因比其他的甜菜碱生物合成和尿素循环基因更加多样化。因此，在蛋白质水平上探索三维结构是否也有很大差异以及酶对L-精氨酸的反应如何不同是非常有趣的。提出了克服哺乳动物和大肠杆菌不溶性和聚集性的策略。线圈NAGS的建议。这包括使用NAGS基因的随机诱变，然后进行GFP筛选和功能测定以鉴定高度可溶性和稳定的突变体。第二种方法使用NAGS与可溶性蛋白质如绿色荧光蛋白（GFP）或麦芽糖结合蛋白（MBP）的融合以增加蛋白质溶解度和稳定性。将产生具有不同性质的不同长度的氨基酸连接体，以检查它们如何影响结晶行为。最终目标是研究来自低等和高等生物的NAGS酶的三维结构，并了解其催化和调节机制，以及与引起高氨血症的遗传缺陷的关系。