Overexpression/crystallization of amino acid transporter

描述（由申请人提供）： 氨基酸是蛋白质和许多其他分子的基本组成部分 在牢房里在中枢神经系统中， 分子。氨基酸的吸收和分泌穿过细胞膜， 由一组称为氨基酸转运蛋白的膜蛋白介导。这些 转运蛋白参与许多重要的生理过程，包括： 小肠吸收上皮的营养摄取、转运 在肾脏近端小管处用于葡萄糖生成和肝再生， 母亲在胎盘处向胎儿提供氨基酸， 突触的神经传递同样重要的是， 与许多病理状况有关，例如胱氨酸尿症， 赖氨酸尿、精神分裂症、局部缺血和可卡因成瘾。 为了了解氨基酸转运蛋白的分子机制， 晶体学产生的三维结构信息， needed.来自哺乳动物生物体的转运蛋白目前不适用于 结晶，由于其天然丰度低，缺乏适当的 过表达系统。细菌氨基酸转运蛋白共享序列 与它们的哺乳动物对应物同源，因此最有可能具有类似的 三维结构。因此，我们计划过度表达，纯化， 使细菌氨基酸转运蛋白从氨基 酸/多胺/胆碱转运蛋白家族。在E. coli中，并纯化至均一。结晶策略始于 筛选来自不同物种的同源物，并通过遗传修饰进行蛋白质修饰 技术.结晶成核将与各种聚乙烯筛选 二醇和pH值。通过调整洗涤剂，可以改善结晶有序性 胶束尺寸和与抑制剂和重金属盐的共结晶。 有序的晶体将使我们能够确定运输结构， 原子分辨率以后结构将揭示氨基酸结合 口袋和底物转运途径，并将使我们能够设计 实验阐明关键残留物在运输中的作用 过程这种结构信息也将揭示结构， 哺乳动物氨基酸转运蛋白的分子机制。

DESCRIPTION (provided by applicant): Amino acids are essential building blocks for proteins and many other molecules in the cell. In the central nervous system, they serve as major signaling molecules. Amino acid uptake and secretion across the cell membrane are mediated by a group of membrane proteins, called amino acid transporters. These transporters are involved in many important physiological processes, including: nutrient uptake at the absorptive epithelia in the small intestine, transport at the kidney proximal tubule for glucogeonesis and hepatic regeneration, supply of amino acids to the fetus by the mother at the placenta, and chemical neurotransmission at the synapse. Equally important, amino acid transporters are associated with numerous pathological conditions, such as cystinuria, lysinuria, schizophrenia, ischemia and cocaine addiction. To understand the molecular mechanism of amino acid transporters, three-dimensional structural information generated by crystallography is needed. Transporters from mammalian organisms are currently not suitable for crystallization, due to their low natural abundance and lack of appropriate overexpression systems. Bacterial amino acid transporters share sequence homology with their mammalian counterparts, and thus most likely have similar three-dimensional structures. We therefore plan to overexpress, purify and crystallize bacterial amino acid transporters from the amino acid/polyamine/choline transporter family. Proteins will be overexpressed in E. coli and purified to homogeneity. Crystallization strategy begins with screening homologues from different species and protein modification by genetic techniques. Crystal nucleation will be screened with various polyethylene glycols and pHs. Crystalline order will be improved by adjusting detergent micellar size and co-crystallization with inhibitors and heavy metal salts. Well-ordered crystals will enable us to determine the transporter structure at atomic resolution later. The structure will reveal the amino acid binding pocket and substrate translocation pathway, and will allow us to design experiments to elucidate the roles that critical residues play in the transport process. Such structural in formation will also shed light on the structure and molecular mechanism for mammalian amino acid transporters.