节点文献
人GOPC蛋白PDZ结构域溶液结构的测定及其与Neuroligin和Frizzled 8 C-末端多肽相互作用的研究
【作者】 李祥；
【作者基本信息】 中国科学技术大学 ， 生化与分子生物学， 2006， 博士
【摘要】 本论文工作的重点是一种高尔基体相关蛋白质——GOPC的PDZ结构域的克隆、表达纯化、溶液结构以及功能研究。我们克隆、表达和纯化了人GOPC蛋白的PDZ结构域；用核磁共振波谱学的方法测定了这个结构域的溶液结构；运用NMR化学扰动实验对人GOPC蛋白的PDZ结构域与Neuroligin和Frizzled 8 C-末端多肽之间的相互作用进行了研究。我们还克隆、表达和纯化了人AREB6蛋白的Homeobox结构域，对该结构域的表达条件进行了初步探讨，并应用光谱学的方法研究该蛋白质的二级结构。论文整体分为三部分。第一部分第一章是对GOPC蛋白生理功能的综述。GOPC是最早发现的定位于高尔基体的PDZ包含蛋白之一，在不同组织中广泛表达。GOPC蛋白在细胞信号调节、细胞自吞噬作用和细胞凋亡途径调控、神经突形成过程的调控等诸多生物学过程中具有重要角色，并能够与诸如TC10，CFTR，frizzled，CALEB／NGC，Golgin-160，β1AR，Neuroligin等许多生物大分子相互作用。GOPC的缺失和突变会导致许多遗传疾病。GOPC基因很可能是一个管家基因。第二章综述了Neuroligin的生物学功能。Neuroligin具有高度保守的C末端序列，是典型的单跨膜蛋白。Neuroligin能与许多蛋白质分子如β-neurexins、突触支架分子(SSCAM)、PSD-95等相互作用，从而在触发突触前膜发育，诱导突触的形成、重塑、成熟，调控兴奋性／抑制性突触的比例等生物学过程中发挥重要作用。Neuroligin的突变会导致某些疾病的发生。论文第二部分利用核磁共振波谱学测定了人GOPC蛋白的PDZ结构域的溶液结构，并对该结构域与Neuroligin和Frizzled 8 C-末端多肽的结合性质进行了研究。编码人GOPC PDZ结构域的基因在大肠杆菌中得到了克隆和表达，利用Ni离子亲和层析法获得可用于核磁实验的重组蛋白质。GOPC蛋白的PDZ结构域具有PDZ结构域家族典型的结构特征：六段反平行的β折叠和两段α螺旋。动力学分析表明，溶液中GOPC蛋白的PDZ结构域在303K时主要以单体形式存在，但在温度降低时有形成聚合体的趋势。利用化学位移扰动实验研究了GOPC蛋白的PDZ结构域与Neuroligin和Frizzled C-末端多肽的结合性质。Neuroligin以及人Frizzled 8 C-末端多肽与GOPC PDZ结构域的作用位点都位于βB和αB螺旋形成的沟中。在NMR的时间尺度上，GOPC PDZ结构域与NeuroliginC-末端多肽的相互作用形成的复合物在整体上属于快速交换的范畴，与人Frizzled 8 C-末端多肽相互作用属于中等强度的相互作用。通过NMR滴定动力学计算出GOPC PDZ结构域与Neuroligin C-末端多肽相互作用的解离常数为260±86μM。我们运用分子动力学模拟的方法，构建了GOPC PDZ结构域与Neuroligin C-末端多肽相互作用的三维结构模型，该模型能很好的解释化学干扰实验的结果。第三部分是关于人AREB6 Homeobox结构域的表达、纯化及二级结构的初步研究。AREB6是一种同时具有锌指和Homeobox的转录因子。人AREB6的Homeobox结构域在大肠杆菌中得到了克隆和表达。我们对其表达和纯化条件进行了研究，并利用圆二色谱(CD)研究了其二级结构。
【Abstract】 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.