Conformational Changes in Glutamate transporters

谷氨酸转运蛋白的构象变化

• 批准号: 8557037
• 负责人: Joseph A Mindell
• 金额: $ 101.58万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557037
• 关键词: Affinity; Alzheimer' s Disease; Bacteria; Biochemical; Biological Models; Chloride Ion; Chlorides; Cut protein; Cysteine; Development; Disease; Electron Spin Resonance Spectroscopy; Energy Transfer; Fluorescence Resonance Energy Transfer; Glutamate Transporter; Glutamates; Goals; Homologous Gene; Knowledge; Lead; Membrane; Methods; Modeling; Movement; Mutagenesis; Neuraxis; Neurons; Neurotransmitters; Play; Process; Protein Binding; Proteins; Proteolysis; Reporting; Role; Scanning; Shapes; Structural Models; Synapses; Therapeutic Agents; Work; extracellular; uptake

This was the sixth year for this project, which is using biochemical and biophysical methods to examine conformational changes related to transport in glutamate transporters. These proteins are of critical importance in the central nervous system, where they play important roles in clearing neurotransmitters from synapses and in shaping the electrical activity of post-synaptic neurons. These transporters have been implicated as playing roles in a variety of diseases, including ALS, Alzheimers disease, and exitotoxicity. It is critical to understand the fundamental mechanisms by which there transporters function because such knowledge could lead to the development of therapeutic agents active against these proteins. We seek to analyze the dynamic movements of the functioning transporter on the way to a detailed understanding of its mechanism. Our approach is to analyze the details of transport in model glutamate transporters obtained from bacteria. These can be expressed and purified in large quantities and are amenable to biophysical methods not available for their mammalian cousins. We previously discovered that the bacterial glutamate transporters display a chloride transport activity which is stoichiometrically uncoupled from glutamate uptake. This chloride transport activity is similar to one which is important in the mammalian transporters and suggests that the bacterial homologs provide an excellent structural model in which to study the process of chloride transport in these proteins. Our studies on conformational dynamics continued this year. We recently reported that a extracellular loop of gltPH must be intact for effective transport. This year we probed the mechanism of this effect in detail and found that when the 34 loop is cut the proteins maintains substrate affinities but maximal transport is significantly reduced. We demonstrated that this effect relates to the activation energy of the substrate translocation step, implicating the loop in the piston like movement of the translocation domain. Our EPR studies have also progressed and reveal that the apo form of GltPh is inward-facing in the membrane and shifts to a more outward form as the protein binds substrate.

这是该项目的第六年，该项目使用生物化学和生物物理方法来研究与谷氨酸转运蛋白转运相关的构象变化。这些蛋白质在中枢神经系统中至关重要，它们在清除突触中的神经递质和塑造突触后神经元的电活动中发挥重要作用。这些转运蛋白已被认为在多种疾病中发挥作用，包括ALS、阿尔茨海默病和兴奋毒性。了解转运蛋白发挥作用的基本机制是至关重要的，因为这些知识可能导致开发对这些蛋白质有活性的治疗剂。我们试图分析的方式，以详细了解其机制的动态运动的功能运输。我们的方法是分析从细菌中获得的模型谷氨酸转运蛋白的运输细节。它们可以大量表达和纯化，并且适用于它们的哺乳动物表亲所不具备的生物物理方法。我们以前发现，细菌谷氨酸转运蛋白显示氯离子转运活性，这是从谷氨酸摄取化学计量解耦。这种氯离子转运活性类似于哺乳动物转运蛋白中的重要活性，并表明细菌同系物提供了一个极好的结构模型，用于研究这些蛋白质中氯离子转运的过程。今年我们继续进行构象动力学的研究。我们最近报道，gltPH的细胞外环必须是完整的有效运输。今年，我们详细探索了这种效应的机制，发现当34环被切断时，蛋白质保持底物亲和力，但最大转运显著降低。我们证明了这种效应与底物易位步骤的活化能有关，暗示了易位结构域的活塞样运动中的环。我们的EPR研究也取得了进展，并揭示了GltPh的载脂蛋白形式在膜中是向内的，并随着蛋白质结合底物而转变为更向外的形式。