Structural Insights Into Arabidopsis Proteins ESP,NSP And NPR4

Thioglucosides (Glucosinolates) is an important metabolic product in cruciferous plants. When the functional factors, such as epithiospecifier protein (ESP), nitrile-specifier protein (NSP) and Thiocyanate-forming protein (TFP) in the presence or absence, it has different hydrolyzates. When the TFP present, it is hydrolyzed into nitrile, epithionitrile and thiocyanate. When the ESP exists, it is hydrolyzed into nitrile and epithionitrile. Nitrile obtained when the NSP present. In order to study the mechanism of glucosinolates metabolism of different products, we studied the structural of Arabidopsis proteins ESP and NSP by means of X-ray.We got 114 conditions of ESP. Finally, we obtained high resolution diffraction data and the crystal phase information about ESP using the crystals under the conditions 0.2 M Sodium chloride,0.1 M HEPES pH7.5,30% PEG400. While crystallization of NSP protein is screening. Structural analysis showed that ESP and TFP which has been resolved have the same structure, but the amino acid residues in the active site are different. Therefore the two proteins have different substrate specificities. We also conducted ESP potential substrates of molecular docking experiments to clarify its substrate binding mode.Another part of this thesis is expression, purification and crystallization of salicylic acid potential receptor protein NPR4. Salicylic acid (SA) is the only major plant hormone for which the receptor has not been firmly identified. The research shows that the NPR1 paralogues NPR3 and NPR4 are SA potential receptors. In the course of our work, we successfully constructed 12 recombinant plasmids of NPR4. We got the high purity proteins about many clones of NPR4. Finally,74 crystallization conditions of NPR4 (1-574aa) were obtained. We mainly optimized MD38-2-21# of all to obtain a good crystal form.Unfortunately its diffraction is low which can not meet the needs of structural analysis.