Vibrational Spectroscopic Investigations of Glutamate Receptor

描述（申请人提供）：谷氨酸和谷氨酸受体之间的特定分子相互作用是这种膜结合蛋白功能变构调节的核心，它是哺乳动物中枢神经系统中主要的兴奋性神经递质受体。最近对载脂蛋白和配体结合形式的谷氨酸受体的分离配体结合域的光谱研究提供了这种相互作用的第一个观点。现在需要的是更详细地了解这些化学相互作用在介导最终调节离子流动的构象变化序列中的作用。我们建议采用两种策略来实现这一目标。首先，使用一组表现出广泛活性的突变体，我们将使用振动光谱识别特定配体蛋白质相互作用的变化，并使用荧光共振能量转移测量将这些变化与配体结合域的裂缝闭合构象变化联系起来。因此，在配体结合区域中确定的变化将通过电生理测量来研究受体功能的变化。其次，我们将监测配体结合域的动力学事件，并将这些事件与受体的功能后果联系起来，即激活和脱敏。对于这种相关性，将使用一组已知可以改变配体结合域的一个或多个动力学步骤的突变体，并将研究对配体结合域结构变化的具体影响以及相应的功能变化。这些平衡和动力学研究将提供对分子水平上的变化的全面理解，这些变化驱动了配体结合域大构象变化的变化，并最终控制了离子通道的功能变化。更重要的是，这种相关性将有助于弥合分离的配体结合结构域与完整受体行为之间的研究差距，并为合理设计旨在调节该重要蛋白功能的药物提供基础，该蛋白已知在包括癫痫和缺血在内的多种神经病理中发挥重要作用。

DESCRIPTION (provided by applicant): The specific molecular interactions between glutamate and the glutamate receptor are central to the allosteric regulation of function in this membrane bound protein, which is main excitatory neurotransmitter receptor in the mammalian central nervous system. Recent spectroscopic investigations of the isolated ligand-binding domain of the glutamate receptor in the apo and ligand bound forms provide a first view of such interactions. What is now needed is a more detailed understanding of the role of these chemical interactions in mediating the sequence of conformational changes that ultimately regulate ion flow. We propose to address this goal using two strategies. First, using a panel of mutants that exhibit a wide range of activities we will identify changes in specific ligand protein interactions using vibrational spectroscopy and correlate these to changes in the cleft closure conformational change in the ligand binding domain using fluorescence resonance energy transfer measurements. The changes thus identified in the ligand binding domain will then be contextualized in terms of changes in receptor function that will be studied by electrophysiological measurements. Secondly, we will monitor the kinetic events in the ligand binding domain and correlate these to the functional consequences in the receptor, i.e., activation and desensitization. For such a correlation, a panel of mutants that are known to alter one or more of the kinetic steps in the ligand binding domain will be used and the specific effects on the structural changes in the ligand binding domain as well as corresponding changes in function will be investigated. These equilibrium and kinetic investigations will provide comprehensive understanding of the changes at the molecular level that drive the changes in the large conformational changes in the ligand binding domain and eventually control the functional changes of the ion channel. More importantly, such correlations will aid in bridging the gap between studies on the isolated ligand-binding domain and the behavior of the intact receptor, and provide a basis for rational design of drugs aimed at modulating the function of this important protein which is known to play an important role in diverse neuropathologies, including epilepsy and ischemia.