Structural Studies of NAGS

描述(由申请人提供)： 这项研究培训申请的目的是提供教学和研究培训经验，使候选人成为一名独立的调查人员。培训的目的是扩大应聘者在分子生物学和分子微生物学方面的专长，以补充他在结晶学方面的知识和经验。因此，其中三名导师拥有分子生物学方面的专业知识，另外两名导师将在蛋白质结构分析的各个方面提供专业知识。这些研究项目旨在了解N-乙酰谷氨酸合成酶(NAGS)的三维结构和功能之间的相关性，该基因是由最近克隆的这种独特的尿素循环酶基因编码的，被认为参与了尿失禁的调节。人们对NAGS蛋白的结构知之甚少。细菌、真菌和哺乳动物的NAGS基因比其他精氨酸生物合成和尿素循环基因更具多样性。因此，在蛋白质水平上探索三维结构是否也非常不同，以及酶对L-精氨酸的不同反应是非常有趣的。提出了一种克服哺乳动物和大肠杆菌NAG的不溶性和聚集性的策略。这包括使用NAGS基因的随机突变，然后进行GFP筛选和功能测定，以鉴定高度可溶和稳定的突变体。第二种方法是将NAG融合成一种可溶性蛋白质，如绿色荧光蛋白(GFP)或麦芽糖结合蛋白(MBP)，以增加蛋白质的溶解度和稳定性。不同长度、不同性质的氨基酸连接物将被生产出来，以考察它们对结晶行为的影响。最终目的是研究低等生物和高等生物NAGS酶的三维结构，了解它们的催化和调节机制，以及与导致高氨血症的遗传缺陷的关系。

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.