Dynamic properties of a glutamate binding domain

 描述（由申请人提供）：离子型谷氨酸受体控制多种正常神经元过程，包括学习和记忆。这些神经递质受体的不适当激活在许多神经退行性疾病的病理学中起作用，特别是中风和癫痫。跨膜拓扑结构的分析已经导致实现，每个亚基是由几个几乎独立的模块。结合谷氨酸的模块，配体结合结构域（LBD）可以在细菌中作为可溶性蛋白大量产生，这使得其结构得以确定。AMPA和红藻氨酸受体的LBD以与完整受体大致相同的亲和力结合激动剂和拮抗剂，并且用作研究结合结构域及其在完整蛋白质的功能中的作用的优良系统。我们以前的工作主要集中在GluA2 LBD（AMPA受体）的结构和动力学上，并使用NMR光谱学和晶体学定义了结合部分激动剂，拮抗剂和变构调节剂后的变化，这反过来又提高了我们对通道激活的结构基础的理解。尽管取得了这一进展，我们知道很少AMPA和红藻氨酸受体，尽管它们的结构相似，有这样不同的动力学特性。我们最近优化了红藻氨酸受体（GluK2）LBD的表达和纯化，以通过NMR光谱进行研究。基于这项工作和我们以前的研究结果描述结合激动剂后氢键和甲基动力学的变化，我们将研究改变GluA2的动力学性质的突变，类似于GluK2，反之亦然。这项工作将采用核磁共振光谱学，结晶学，等温滴定量热法，全细胞记录，和热变性。这将使我们能够确定结合域如何控制动力学特性，从而为红藻氨酸和AMPA受体的不同生理特性塑造突触电流。该项目的第二部分将研究部分激动剂激活谷氨酸受体的机制。我们以前的工作与AMPA受体表明，部分激动剂可以激活通道诱导过渡到构象的LBD的特点是一个完整的叶关闭，但较低的概率比完全激动剂。红藻氨酸受体具有更广泛的部分激动剂，其可以通过不同的机制起作用。红藻氨酸受体中的部分激动剂的表征，结合高场NMR光谱学，旨在研究低概率的构象，将使我们能够剖析部分激动剂结合的机制，并将其与我们以前的结果从单通道记录。这些研究将使用一系列生物物理技术，旨在了解AMPA和红藻氨酸受体的动力学特性的结构基础。这将为开发针对这些必需神经递质受体的药物提供重要信息。

 DESCRIPTION (provided by applicant): Ionotropic glutamate receptors control a wide variety of normal neuronal processes including learning and memory. Inappropriate activation of these neurotransmitter receptors plays a role in the pathology of a number of neurodegenerative diseases, notably stroke and epilepsy. Analysis of the transmembrane topology has led to the realization that each subunit is made of several nearly independent modules. The module that binds glutamate, the ligand binding domain (LBD) can be produced in large quantities in bacteria as a soluble protein, which has allowed its structure to be determined. The LBDs of AMPA and kainate receptors bind agonists and antagonists with approximately the same affinity as the intact receptor and serve as an excellent system for studying the binding domain and its role in the function of the intact protein. Our previous work has focused on the structure and dynamics of the GluA2 LBD (AMPA receptor) and has defined the changes upon binding partial agonists, antagonists and allosteric regulators using NMR spectroscopy and crystallography, which in turn has improved our understanding of the structural basis of channel activation. Despite this progress, we know little about how AMPA and kainate receptors, despite their structural similarity, have such divergent kinetic properties. We have recently optimized the expression and purification of a kainate receptor (GluK2) LBD for study by NMR spectroscopy. Building upon this work and our previous results describing changes in hydrogen bonding and methyl group dynamics upon binding agonists, we will study mutations that alter the kinetic properties of GluA2 to resemble those of GluK2 and vice versa. This work will employ NMR spectroscopy, crystallography, isothermal titration calorimetry, whole cell recording, and thermal denaturation. This will allow us to determine how the binding domain controls kinetic properties and thus shapes synaptic currents for the different physiological properties of kainate and AMPA receptors. The second part of the project will investigate the mechanisms by which partial agonists activate glutamate receptors. Our previous work with AMPA receptors indicated that partial agonists may activate the channel by inducing transitions to a conformation of the LBD characterized by a full lobe closure, but with lower probability than full agonists. Kainate receptors have a wider range of partial agonists that may operate by different mechanisms. The characterization of partial agonism in kainate receptors, combined with high field NMR spectroscopy designed to study conformations of low probability, will allow us to dissect the mechanism of partial agonist binding and relate it to our previous results from single channel recording. These studies will use a range of biophysical techniques designed to understand the structural basis of the kinetic properties of AMPA and kainate receptors. This will provide essential information for development of drugs directed toward these essential neurotransmitter receptors.