Study Of The Dynamic Of The Inner Active Center Loop Of E1 Component Of E.coli Pyruvate Dehydrogenase Complex In The Catalytic Event And The Hexadecylation Of The N-terminal Peptide Of Human Postsynaptic Density-95

Nuclear magnetic resonance(NMR) and organic modification of protein are two important methods in the studies of protein structure and function. NMR can be directly used to study the dynamics of proteins, while the structure of protein can often be deduced from the difference between original protein and modified protein. In our work, the two analysis methods were employed to study the relationship of structure and function of two kinds of protein, and some useful results were obtained.The thiamin diphosphate(ThDP)-dependent E1 component(pyruvate dehydrogenase) of pyruvate dehydrogenase complex(PDC) of E. coli plays an important role in the catalytic events of PDC. With the synergy of thiamine pyrophosphate(ThDP), it catalyzes the decarboxylation of pyruvic acid to acetylatedlipoic acid, and offers the product to the next catalytic step. The X-ray analysis of the cocrystal of E1 and ThDP, E1 and thiamin thiazolonediphosphate(ThTDP), E1 and α-phosphono-lactylthiamindiphosphate(PLTh DP) and the crystal of apo-E1 showed that the dynamic of the inner active center loop of E1 was closely related to the catalytic process. To study the dynamic structure of the inner active center loop of E1, we expressed four kinds of mutants, pET-28a-E1-H407 A,pET-28a-E1-K403 P,pET-28a-E1-k410 P and pET-28a-E1-K403P/K410 P in the of vector pET-28a-E1 by PCR-driven overlop extension. The corresponding amino acids of the mutant sites in the four mutants are considered very relevant to the activity of PDC. The prolin residue is considered as rigid amino acid in structure and may make the structure of inner active center loop more rigid. Then, we expressed and purified these proteins. When expressing the vector pET-28A-E1 and four kinds of mutants, we inserted prescission protease digestion sites. The His-tag were removed by adding the prescission protease to protein solutions to avoid the side effect for subsequent experiments.15NH4 Cl was used in the express of proteins. After obtained the proteins, NMR was used to get the 1H-15 N HSQC spectra of them. So far, we have got the 1H-15 N HSQC spectra of E1, H407 A mutant, E1-ThDP and H407A-ThDP. The comparison of these spectra pointed out that the position of the cross peak of H407 site. By comparing the different chemical shift values, we confirme the direct interaction between H407 site and ThDP. In order to reveal more information about the dynamic changes in the inner active center loop in E1 catalysis, the effect of PLThDP on His407 and the effect of ThDP/PLThDP on K403/K410 will be studied via 1H-15 N HSQC in our group.As a member of membrance associated guaylate kinase, postsynaptic density-95(PSD-95) plays a very important role in the transmission of nerve signals. Previous studies have also shown that PSD-95 is crucial in the tramission of N-methyl-D-aspartate signals. PSD-95 is also involved in many life processes such as monitoring the plasticity of visual cortical neurons in the visual development sensitive period and signal transmission in spinal cord development and sipnal cord injury. The study of interaction between PSD-95 and cyclin-dependent kinase-like 5(CDKL-5) further indicated that only the palmitoylation state of PSD-95 can perform its function, and the binding region with CDK-L5 is mainly the 1-21 N-terminal amino acids. The corresponding peptide was synthesized in our work. Then we designed a series of experiments to hexadecylate the 21 peptide in vitro, which wound obtain the analog of palmitoylated PSD-95 21 peptide and wound develop a new method for PSD-95 research.Our experiment contains two steps. In the first step, the 21 peptide was treated with DL-Dithiothreitol(DDT) to afford sulfhydryl group from the disulfide bond in the peptides, then the product was purified with HPLC and freeze-dried to yield the reduced form of 21 peptide. The second step is employing the thiol-ene click chemistry to conjugate the 1-hexadecene to the 21 peptide. After purification and freeze drying, the success of the 1-hexadecene modification was confirmed via contrasting the sulfhydryl group concentration in the original 21 peptide and reduced 21 peptide, as well as the mass spectrometry.The successful modification of the 21 peptide with 1-hexadecene is a crucial preliminary work of the in vitro palmitoylation of PSD-95, which is important for the functional mechanism research of PSD-95. At the same time, the palmitoylation site of PSD-95 could be regard as a targeting site of drug design of Alzheimer’s Disease and Rett’s syndrome.