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.
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