People Gopc Protein Pdz Domain Solution Structure Of Neuroligin And Frizzled - 8 C-terminal Peptide Interaction

Our work focuses on the clone, expression, purification and structural and functional studies of PDZ domain of GOPC (Golgi-associated PDZ-and coiled-coil motif-containing protein) associated with the Golgi apparatus. The PDZ domain of human GOPC has been successfully cloned, expressed and purified. The solution structure of this PDZ domain has been determined by NMR spectroscopy. Using NMR chemical shift perturbation experiments, we investigate the binding characteristics of PDZ domain to the C-terminal peptides of Neuroligin and Frizzled 8 in vitro. Homeobox domain of humain AREB6 (hAREB6 Homeobox) has been successfully cloned, expressed and purified. We have explored the expression conditions and the secondary structure of hAREB6 Homeobox.There are three sections in our thesis. The first section introduces the reviews of the biological background of GOPC and neuroligin. As one of the first reported PDZ containing proteins localized at the Golgi apparatus, GOPC is expressed extendedly in many tissues and maybe a housekeeping gene. GOPC plays important roles including regulating signal, modulating pathways of apoptosis and autophagy, and controlling the growth of neurite, etc., which can interact with many molecules such as TC10, CFTR, frizzled, CALEB/NGC, Golgin-160,β1AR, Neuroligin. Many genetic diseases in humans are involved in the deletion and mutation of GOPC. Neuroligin is a postsynaptic cell-adhesion molecule of synapses which contains five distinct domains: a N-terminal sequence, a acetylcholinesterase homology domain, a linker domain, a single transmembrane domain, and a short intracellular sequence with a highly conserved C-terminus. The interactions between neuroligin and its binding partner, such asβ-neurexin, SSCAM, PSD-95, could induce formation, reconstruct, maturation of the synapse. Neuroligin control the functional balance of excitatory and inhibitory synapses and regulate the excitatory/inhibitory synaptic ratio through interaction with its postsynaptic binding partner.In section II , the solution structure of human GOPC PDZ domain was determined by NMR spectroscope and the binding characteristics of this domain with the C-terminal peptide of Neuroligin (Nlg (817-823)), and that of Frizzled 8 (Fz (687-694)) were studied. Recombinant PDZ domain of GOPC was cloned, expressed in E.coli and purified by Ni-chelating column. The NMR-derived tertiary structure of the GOPC PDZ domain is a canonical PDZ fold that contains twoα-helices and sixβ-strands. The dynamic properties indicate that the GOPC PDZ domain might be not aggregated or that the aggregation is very little at 303 K, and that there is a balance between monomer and aggregation of the GOPC PDZ domain and the shift of balance depends on the temperature. The binding properties of the GOPC PDZ domain with Nlg (817-823) and Fz (687-694) were characterized by NMR chemical shift perturbation experiments. The results indicate that both of them extend in the cleft between theβB strand and theαB helix, as most PDZ-binding ligands do. The ensemble of the binary complexes with Nlg (817-823) and Fz (687-694) belongs to fast exchange and intermediate exchange on the NMR timescale, respectively. The dissociation constants of the interaction between the GOPC PDZ domain and Nlg (817-823) calculated according to titrating dynamics is 260±86μM. The 3D model of GOPC PDZ-Nlg (817-823) was built up by molecular dynamics simulations, which can explain the results of chemical shift perturbation experiments.Section III introduces the work of Homeobox domain of human AREB6 (hAREB6 Homeobox). AREB6 is a transcription factor which contains zinc finger and homeobox domain. hAREB6 Homeobox is cloned, expressed in E.coli and purified by Ni-chelating column. The conditions of expression and purification of hAREB6 Homeobox were studied, and the secondary structure of this domain was determined by circular dichroism.