Spectroscopic Investigations of Glutamate Receptor

描述（由申请人提供）：谷氨酸和谷氨酸受体之间的特异性分子相互作用对于该膜结合蛋白的功能变构调节至关重要，该膜结合蛋白是哺乳动物中枢神经系统中的主要兴奋性神经递质受体。最近的光谱研究分离的配体结合结构域的谷氨酸受体在载脂蛋白和配体结合的形式提供了第一个这样的相互作用。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的构象变化序列中的作用。我们建议使用两种策略来实现这一目标。首先，使用一组突变体，表现出广泛的活动，我们将确定在特定的配体蛋白质相互作用的变化，使用振动光谱和相关的这些变化的裂缝闭合构象变化的配体结合结构域，使用荧光共振能量转移测量。因此，在配体结合结构域中鉴定的变化然后将根据将通过电生理学测量研究的受体功能的变化进行情境化。其次，我们将监测配体结合结构域中的动力学事件，并将其与受体中的功能结果相关联，即，激活和脱敏。对于这种相关性，将使用已知改变配体结合结构域中的一个或多个动力学步骤的一组突变体，并研究对配体结合结构域中的结构变化以及相应的功能变化的特定影响。这些平衡和动力学研究将提供在分子水平上的变化，驱动配体结合结构域中的大的构象变化的变化，并最终控制离子通道的功能变化的全面理解。更重要的是，这种相关性将有助于弥合对分离的配体结合结构域和完整受体的行为的研究之间的差距，并为旨在调节这种重要蛋白质的功能的药物的合理设计提供基础，已知这种重要蛋白质在包括癫痫和缺血在内的多种神经病理学中发挥重要作用。

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.