Selective Detection Of Protein Side Chain NH2 Signals By NMR

Three-dimensional (3D) structure of a protein is helpful in understanding its function, and is the base for drugs screening and designing. X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) spectroscopy are the two techniques for determining the protein structure. XRD is a most efficient and fast way to obtain high-resolution protein structure. About 85% of the structures in the protein data bank (PDB) were determined using XRD approach. The disadvantage of XRD is the requirement of single crystals. NMR is the only method available for the determination of the 3D protein structure in solution. In addition, NMR can provide information about the protein dynamics and interaction. Although NMR measurements, data processing and structure refinements take longer time than XRD, it dose not need single-crystal and works in the solution that is close to the native state of protein.In this thesis, apply a modified HSQC experiment to identify protein side chain NH2 signals. Protein PDZ7 is used to test the modified experiment. The backbone NMR resonances of PDZ7 were assigned again based on our experiments for building-up the experience of protein structure determination. In addition, new developments in NMR techniques for protein structure determination were reviewed. In the first Chapter, the basic NMR principles were introduced with special attentions to the methods (NMR pulse sequences) used in protein structure determination.In the second Chapter, the strategies for protein NMR structure evaluation and calculation were introduce.In the third Chapter, the modified HSQC experiment for selective detection of side-chain NH2 was described. The method was based on the conventional HSQC experiment by adding an extra delay (1/21JHN) before t1 period. During this delay time, the processing of 1JHN caused the NH signal undetectable, while the signal from NH2 groups remained unchanged. This resulted in a spectrum containing the correlation of the side chain NH2. PDZ7 protein was used to test the modified experiment. In addition, the backbone resonances of PDZ7 were assigned based on our experiments to gain the experience for protein structure determination.