Overexpression and crystallization of amino acid transp*

氨基酸 transp* 的过表达和结晶

• 批准号: 6524654
• 负责人: DANENG WANG
• 金额: $ 16.85万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524654
• 关键词: X ray crystallography; aminoacid; bacterial genetics; bacterial proteins; crystallization; gamma aminobutyrate; gene expression; membrane transport proteins; molecular cloning; phenylalanine; protein structure; recombinant proteins; synchrotrons

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.

描述（由申请人提供）： 氨基酸是蛋白质和许多其他分子的重要组成部分 在细胞中。在中枢神经系统中，它们充当主要信号传导 分子。跨细胞膜的氨基酸摄取和分泌 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： 小肠吸收上皮细胞对营养物质的吸收、运输 在肾近端小管进行糖生成和肝再生， 母亲通过胎盘向胎儿供应氨基酸，以及化学物质 突触处的神经传递。同样重要的是氨基酸转运蛋白 与许多病理状况有关，例如胱氨酸尿症， 赖氨酸尿症、精神分裂症、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是 需要。来自哺乳动物的转运蛋白目前不适合 结晶，由于它们的自然丰度低且缺乏适当的 过度表达系统。细菌氨基酸转运蛋白共享序列 与其哺乳动物对应物具有同源性，因此很可能具有相似的 三维结构。因此，我们计划过度表达、纯化和 从氨基酸中结晶细菌氨基酸转运蛋白 酸/多胺/胆碱转运蛋白家族。蛋白质将在 E 中过度表达。 大肠杆菌并纯化至均质。结晶策略始于 筛选不同物种的同源物并通过遗传修饰蛋白质 技术。晶体成核会用各种聚乙烯进行筛选 乙二醇和 pH 值。通过调整洗涤剂可以改善晶序 胶束大小以及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转运蛋白的结构 原子分辨率稍后。该结构将揭示氨基酸结合 口袋和底物易位途径，并使我们能够设计 阐明关键残基在运输中所起作用的实验 过程。这种结构信息也将阐明结构和 哺乳动物氨基酸转运蛋白的分子机制。